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Electronic Theses and Dissertations

2011

DEVELOPMENT AND VALIDATION OF AFLOW DEVICE TO STUDY PLATELETFUNCTION IN VITRO AND ELUCIDATINGTHE ROLE OF THYMOSIN β 4 IN VARIOUSPHYSIOLOGICAL PROCESSESHarmanpreet KaurUniversity of Windsor

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Recommended CitationKaur, Harmanpreet, "DEVELOPMENT AND VALIDATION OF A FLOW DEVICE TO STUDY PLATELET FUNCTION INVITRO AND ELUCIDATING THE ROLE OF THYMOSIN β 4 IN VARIOUS PHYSIOLOGICAL PROCESSES" (2011).Electronic Theses and Dissertations. 393.https://scholar.uwindsor.ca/etd/393

DEVELOPMENT AND VALIDATION OF A FLOW

DEVICE TO STUDY PLATELET FUNCTION IN

VITRO AND ELUCIDATING THE ROLE OF

THYMOSIN β 4 IN VARIOUS PHYSIOLOGICAL

PROCESSES

By

Harmanpreet Kaur

A Dissertation

Submitted to the Faculty of Graduate Studies

through the Department of Chemistry and Biochemistry

in Partial Fulfillment of the Requirements for

the Degree of Doctor of Philosophy at the

University of Windsor

Windsor, Ontario, Canada

2011

© 2011 Harmanpreet Kaur

ii

Development and validation of a flow device to study platelet function in

vitro and elucidating the role of thymosin beta 4 in various physiological

processes

by

Harmanpreet Kaur

APPROVED BY:

S. Rafferty, External Examiner

Trent University

R. Carriveau, Departmental External

Department of Civil and Environmental Engineering

P. Vacratsis, Departmental Internal

Department of Chemistry & Biochemistry

S. Ananvoranich, Departmental Internal

Department of Chemistry & Biochemistry

B. Mutus, Advisor

Department of Chemistry & Biochemistry

Dr. M Ahmadi, Chair of Defense

Department of Electrical & Computer Engineering

October 2011

iii

DECLARATION OF CO-AUTHORSHIP / PREVIOUS PUBLICATION

I. Declaration of Co-Authorship

I hereby declare that this thesis incorporates material that is the result of joint research,

as follows:

This thesis incorporates the outcome of research efforts undertaken in the supervision of

Dr. Bulent Mutus. In all cases experimental design, execution, data analysis,

interpretation, and manuscript preparation were performed by the author.

I am aware of the University of Windsor Senate Policy on Authorship and I certify that I

have properly acknowledged the contribution of other researchers to my thesis, and have

obtained written permission from each of the co-authors to include the above materials in

my thesis.

I certify that, with the above qualification, this thesis, and the research to which it refers,

is the product of my own work.

II. Declaration of Previous Publication

This thesis includes 3 original papers that have been previously published/submitted for

publication in peer reviewed journals, as follows:

Thesis Chapter Publication Title and Full Citation Publication Status

Chapter 2 Development of flow device to study effects of

shear stress on endothelial cells and its

applications

Accepted 2011

Chapter 4 Whole blood, flow-chamber studies in real-time

indicate a biphasic role for thymosin

β-4 in platelet adhesion.

Published 2010

Chapter 5 Thymosin β-4 alleviates endoplasmic reticulum

stress in retinal pigment epithelial cells.

To be submitted

I certify that I have obtained permission from the copyright owner(s) to include the above

published material(s) in my thesis. I certify that the above material describes work

completed during my registration as graduate student at the University of Windsor. I

declare that, to the best of my knowledge, my thesis does not infringe upon anyone‘s

copyright nor violate any proprietary rights and that any ideas, techniques, quotations, or

iv

any other material from the work of other people included in my thesis, published or

otherwise, are fully acknowledged in accordance with the standard referencing practices.

Furthermore, to the extent that I have included copyrighted material that surpasses the

bounds of fair dealing within the meaning of the Canada Copyright Act, I certify that I

have obtained a written permission from the copyright owner(s) to include such

material(s) in my thesis. I declare that this is a true copy of my thesis, including any final

revisions, as approved by my thesis committee and the Graduate Studies office, and that

this thesis has not been submitted for a higher degree to any other University or

Institution.

v

ABSTRACT

Parallel plate flow chambers, simulating in vivo fluid shear stress, provide a real

time insight into the dynamic process of platelet aggregation and investigation of

endothelial cell response to shear stress. This thesis describes the design and validation of

– 1) A simple parallel plate flow chamber to study effects of shear stress on endothelial

cells. This flow chamber is easy to use, inexpensive and fast to manufacture as compared

to the flow devices reported previously. Moreover, it minimizes the number of cells and

solution volumes to be used. It can be used as an effective in vitro system to study the

effects of fluid shear stress on the structure and function of endothelial cells. 2) A four

channel cylindrical flow device, constructed out of polydimethylsiloxane (PDMS) on

microscope coverslips, to study platelet aggregation under in vivo like conditions. The

novel aspect of this flow device is the surface chemistry we have devised for the facile

patterning of immobilized proteins (fibrinogen and collagen) onto polydimethylsiloxane

surfaces. The flow method introduced here was employed to determine the effect of

thymosin β 4, a G-actin sequestering peptide, on the deposition of ADP-activated platelets

onto fibrinogen cross-linked flow chambers. Platelets carry and release large amounts of

thymosin β 4. Yet the role of thymosin β 4 on platelet thrombus formation has not been

fully investigated. We demonstrate that thymosin β 4 has a dual role in platelet

aggregation. Our results show that at low doses thymosin β 4 promotes platelet deposition

and aggregation by yet unknown mechanism. However, platelet adhesion to fibrinogen is

inhibited at high concentrations of thymosin β 4.

Exogenous thymosin β 4 has also been reported to promote wound healing,

inflammation reduction and protection of human cornea epithelial cells against oxidative

damage. Herein, we show that thymosin β 4 can also assuage endoplasmic reticulum

stress in retinal pigment epithelium cells (RPE). Thymosin β 4 pre treatment before

introducing endoplasmic reticulum stress decreases ROS, cholesterol levels and nuclear

translocation of NFκB in RPE.

In conclusion, this body of work demonstrates the utility of flow devices to study

platelet function and effects of shear stress in endothelial cells under in vivo like

conditions. This work also reveals the role of thymosin β 4 in platelet function and

alleviating ER stress in RPE, both of which might of considerable therapeutic relevance.

vi

Dedicated to my parents and my husband for their tremendous love and support

vii

ACKNOWLEDGEMENTS

It is with profound gratitude and appreciation that I acknowledge the able guidance of my

revered supervisor, Dr. Bulent Mutus. I owe him a lot for his valuable guidance, concern,

constructive criticism and thought provoking discussions at every step. I would also like

to thank my committee members, Dr. Srinart Ananvoranich and Dr. Otis Vacratsis and for

their insightful comments and encouragement.

I am highly obliged to our collaborator and my committee member, Dr. Rupp

Carriveau for designing the flow chamber and for his guidance and valuable suggestions

in these studies. I am also grateful to Dr. Gabriel Sosne for providing thymosin beta 4 for

my work.

I would like to thank all the past and current members of Mutus lab over these

years: Vasantha, Adam, Bei, Rebecca, Artur, Suzie, Rebecca, Christine, Ryan, Ruchi,

Arun, Inga and Shane. My sincere thanks go to Marlene for her support and help. I must

also acknowledge all the faculty and staff of the department for their help and assistance.

My deepest appreciation goes to my friend, Tanreet for always being there for me

and supporting me all these years. Finally, I would like to thank my husband and my

family for their unrelenting support, encouragement and understanding. Without their

support, it would have been impossible for me to complete this work.

viii

TABLE OF CONTENTS

DECLARATION OF CO-AUTHORSHIP/PREVIOUS PUBLICATION iii

ABSTRACT v

DEDICATION vi

ACKNOWLEDGEMENTS vii

LIST OF TABLES xi

LIST OF FIGURES xii

LIST OF ABBREVIATIONS xiv

I. CHAPTER 1 1

General Introduction

1. PLATELETS 2

1.1 Platelet receptors and signalling pathways 3

1.2 Major proteins involved in thrombosis 11

1.3 Methods to study platelet function 12

2. SHEAR STRESS 17

1.1 Role of shear stress in platelet adhesion and aggregation 17

1.2 Endothelial cell responses to shear stress 19

1.3 Shear stress sensors 20

1.4 Shear stress, endothelial dysfunction and atherosclerosis 22

1.5 Parallel plate flow chambers 24

ix

3. THYMOSIN BETA 4 25

3.1 Structure and properties 25

3.2 Biological functions 26

4. RETINAL PIGMENT EPITHELIUM 30

4.1 RPE Functions 31

4.2 RPE ageing and diseases 34

REFERENCES 37

II. CHAPTER 2 49

Development of flow devices to study effects of shear stress on

endothelial cells and its applications

Introduction 50

Experimental Methods 52

Results 55

Discussion 65

Conclusions 66

References 67

III. CHAPTER 3 69

Real-time kinetic analysis of platelet function in a flow chamber with

matrix proteins covalently attached onto a polydimethylsiloxane

surface.

Introduction 70

Experimental Methods 72

Results 75

Discussion 90

Conclusions 91

References 92

x

IV. CHAPTER 4 93

Whole blood, flow-chamber studies in real-time indicate a biphasic

role for thymosin β-4 in platelet adhesion

Introduction 94

Experimental Methods 97

Results 100

Discussion 111

Conclusions 113

References 114

V. CHAPTER 5 116

Thymsoin β 4 alleviates endoplasmic reticulum stress in retinal

pigment epithelial cells

Introduction 117

Experimental Methods 119

Results 123

Discussion 137

Conclusions 138

References 139

VI. CHAPTER 6

General Discussion 141

References 146

APPENDIX 148

VITA AUCTORIS 154

xi

LIST OF TABLES

CHAPTER 1

Table 1 – Platelet membrane receptors and their ligands. 3

CHAPTER 3

Table 1 – Devices available to study platelet aggregation in vitro. 70

Table 2 – Total number of normal and type 2 diabetic (T2D) platelets ± SD 88

attached to fibrinogen (Fib) and collagen (Coll).

Table 3 – First order rate constants calculated from the kinetic plots of 89

the platelet adhesion data.

xii

LIST OF FIGURES

CHAPTER 1

General Introduction

Figure 1. Platelet receptors and ligand interactions. 4

Figure 2. Platelet signalling through GP VI and GP Ib/IX/V. 6

Figure 3. Integrin activation. 8

Figure 4. ADP receptors and downstream signalling. 10

Figure 5. Cone and plate viscometer. 14

Figure 6. Perfusion chamber. 16

Figure 7. Mechanotransduction of endothelial shear stress. 21

Figure 8. Effects of disturbed shear rates on endothelial cell function. 23

Figure 9. Thymosin β4 and blood coagulation. 28

Figure 10. Retinal pigment epithelium. 31

CHAPTER 2

Figure 1. Schematic diagram of the parallel plate flow chamber. 56

Figure 2. Velocity gradient template. 59

Figure 3. Effect of shear stress on morphology of bovine endothelial cells. 60

Figure 4. Nitric oxide production and eNOS phosphorylation in response 62

to shear stress.

Figure 5. Shear stress upregulates caveolin-1 expression in bovine endothelial 64

cells

CHAPTER 3

Figure 1. Flow chamber geometry and immobilization chemistry. 75

Figure 2. Velocity profile. 77

Figure 3. Kinetic data for aminolysis of DSS. 78

Figure 4. Variations in time points at which fibrinogen/collagen is added 80

to DSS affects immobilization.

Figure 5. SDS washing of FITC-fibrinogen 82

Figure 6. Effect of bodipy labelling on platelet aggregation. 83

xiii

Figure 7. Comparison of platelet aggregation in normal and type 2 85

diabetic patients.

Figure 8. Kinetic plots of platelets adhered to immobilized fibrinogen 87

and collagen over time.

CHAPTER 4

Figure 1. Location and activity of different peptides derived from Tβ4. 95

Figure 2. Flow chamber geometry and immobilization chemistry. 100

Figure 3. Demonstration of the Otsu Multi-Thresholding ImageJ plugin 102

to exclude non- deposited, transiently associated platelets

from being counted in the deposition data.

Figure 4. Kinetic plots obtained from particle counts of Otsu Multi Threshold 104

images for platelets deposited onto immobilized BSA, fibrinogen and

collagen

Figure 5. The effect of Tβ4-dose on the rate and density of platelet deposition. 107

Figure 6. Binding of eosin-Tβ4 to fibrinogen immobilized on PDMS. 110

CHAPTER 5

Figure 1. Detection of apoptosis and cell viability in ARPE19 cells treated 124

with palmitate with or without Tβ4.

Figure 2. Expression of ER stress markers, Grp78 and PDI, in ARPE19 125

cells treated with palmitate in the presence or absence of Tβ4.

Figure 3. Effect of Tβ4 pretreatment on cholesterol in retinal pigment 127

epithelial cells under ER stress.

Figure 4. Effect of Tβ4 pretreatment on intracellular ROS production 130

in ER stress induced retinal pigment epithelial cells.

Figure 5. Effect of Tβ4 on nitric oxide production, eNOS expression 132

and phosphorylation in retinal pigment epithelial cells under ER stress

Figure 6. NFκB localization in response to ER stress and Tβ4 pretreatment. 135

xiv

LIST OF ABBREVIATIONS

Abbreviation Definition

ACD Acid Citrate Dextrose

ADP Adenosine diphosphate

AMD Age related macular degeneration

APTMES/AS Aminopropyltrimethoxysilane

ATP Adenosine triphosphate

BAEC Bovine aortic endothelial cells

BCA Bicinchoninic acid

bFGF basic Fibroblast growth factor

BSA Bovine serum albumin

Ca2+

Calcium

CalDAG-GEFI Diacylglycerol-regulated guanine nucleotide

exchange factor I

CFD Computational fluid dynamics

Coll Collagen

DAF-DA 4,5-diaminofluorescein diacetate

DAG Diacylglycerol

DHA Docosahexaenoic acid

DNA Deoxyribonucleic acid

DR Diabetic retinopathy

DSS Disuccinimidyl suberate

DTT Dithiothreitol

ECM Extracellular matrix

EDTA Ethylenediaminetetraacetic acid

EITC Eosin-isothiocyanate

eNOS endothelial Nitric oxide synthase

ER Endoplasmic reticulum

ERS Endoplasmic reticulum stress

FAK Focal adhesion kinase

xv

Fas-L Fas ligand

FcRγ Fc receptor gamma

Fib Fibrinogen

FITC Fluorescein-isothiocyanate

GLUT Glucose transporter isoform

GP Glycoprotein

GPO Gly– Pro–Hyp

H2DCFDA 2′,7′-dichlorodihydrofluorescein

HEPES (4(2-hydroxyethyl)-1-piperazineethanesulfonic acid)

HUVEC Human umbilical vein endothelial cells

ICAM-1 Intercellular adhesion molecule-1

Ig Immunoglobin

IGF Insulin like growth factor

IL-1 Interleukin-1

IP3 Inositol 1, 4, 5 triphosphate

IPM Interphotoreceptor matrix

IRBP Interphotoreceptor retinoid binding protein

ITAM Immunoreceptor tyrosine based activation motif

KC Keratinocyte chemoattractant

LDL Low density lipoproteins

LEDGF Lens epithelium derived growth factor

LPS Lipopolysaccharide

MCP-1 Monocyte chemoattractant protein-1

MIP 2 Macrophage inflammatory protein 2

NFκB Nuclear factor kappa B

NHS N-hydroxysulfosuccinimide

NO Nitric oxide

NSMase 2 Neutral sphingomyelinase 2

PBS Phosphate buffered saline

PDGF Platelet derived growth factor

PDGF Platelet derived growth factor

xvi

PDI Protein disulfide isomerase

PDMS Polydimethylsiloxane

PECAM-1 Platelet endothelial cell adhesion molecule 1

PEDF Pigment epithelium derived factor

PF-4 Platelet factor-4

PFO-D4-GFP Perfringolysin-domain 4-green fluorescent protein

PI3K Phosphoinositide 3 kinase

PIP2 Phosphatidyl inositol 4, 5 bisphosphate

PKC Protein kinase C

PLCγ2 Phospholipase C gamma 2

PPFC Parallel-plate flow chambers

PVDF Polyvinylidene fluoride

ROS Reactive oxygen species

RPE Retinal pigment epithelium

SD Standard deviation

SDS Sodium dodecyl sulphate

SIPA Shear induced platelet aggregation

SOD Superoxide dismutase

SREBP Sterol regulatory elements binding proteins

SSPE Shear stress responsive elements

STIM1 Stromal interaction molecule 1

T2D Type 2 diabetic

TNF α Tumor necrosis factor α

Tβ4 Thymosin β 4

UPR Unfolded protein response

UVB Ultraviolet B

VCAM-1 Vascular cell adhesion molecule-1

VEGF Vascular endothelial growth factor

vWF von Willebrand factor

1

CHAPTER – 1

General Introduction

2

1. Platelets

Hemostasis is a co-ordinated event of various cellular and biochemical interactions,

which involves the arrest of bleeding, formation of platelet aggregates and hence wound

healing. Blood platelets play a very important role in every aspect of hemostasis. Platelets

are small (2 to 4 µm in diameter), anuclear and disc-shaped and colourless cellular

structures with a large number of secretory granules. These cells are derived from mature

megakaryocytes via a process called thrombopoiesis. In the circulation of normal human

beings, the number of platelets is in the range of 150,000 to 350,000 per µL. Their

average life span is 6-10 days, after which they are destroyed by phagocytosis in the

spleen.

Platelets have an important role in blood coagulation. Upon vascular injury,

platelets are activated by subendothelial adhesive proteins like collagen and by a wide

variety of soluble agonists including ADP, thrombaxane A2, thrombin and serotonin.

These agonists induce signalling pathways by binding to their respective receptors on

platelets, which leads to various signalling events such as platelet cytoskeletal changes

and granule secretion. Upon activation by the agonists, platelets change their shape and

adhere to newly exposed subendothelial tissues. The contents of the secretory granules (α

granules and dense granules) are released at the site of vascular damage, which play an

important role in haemostasis and thrombus formation (Zucker et al 1985). Platelet shape

change and aggregation are of central importance for the formation of platelet thrombi

and subsequently wound healing. Platelet plug formation at the site of injury occurs in

three stages.

a) Platelet adhesion – Platelets detect vascular damage and adhere to the exposed

subendothelium forming a monolayer of activated platelets.

b) Platelet release reaction – The platelets release active substances that are stored

in their secretory granules. Serotonin, ADP, ATP and calcium are released by

dense granules. Lysosomes release hydrolytic enzymes and α granules release

platelet factor 4 (PF-4), beta thromboglobulin, platelet- derived growth factor

(PDGF), fibrinogen, fibronectin, von Willebrand factor and albumin. The agonists

released by the attached platelets activate and recruit more platelets.

3

c) Platelet aggregation – The recruited platelets aggregate with those already bound

and form a platelet plug, which serves as a surface for fibrin deposition. The

platelet plug formed is stabilized, eventually leading to wound healing.

1.1 Platelet receptors and signalling pathways

Platelet adhesion and aggregation on the exposed extracellular matrix (ECM) requires the

coordinated interaction of different platelet surface receptors with adhesive

macromolecules. Platelet receptors play a key role in regulating the cascade of events

whereby circulating resting platelets make the rapid transition to an adhered, activated

and aggregated thrombus. The mutation or absence of the receptors or binding proteins

can affect the ability of the platelets to respond to vascular injury.

A wide variety of transmembrane receptors covers the platelet membrane,

including many integrins (αIIbβ3, α2β1), G-protein coupled seven transmembrane receptors

(GPCR) (PAR-1 and PAR-4) thrombin receptors, P2Y1 and P2Y12 receptors and proteins

belonging to the immunoglobulin superfamily (GP VI). Each of these receptors is capable

of binding one or more ligand. Platelet receptors have a prominent role in the hemostatic

function of platelets, allowing specific interactions and functional responses of vascular

adhesive proteins and of soluble platelet agonists. Platelet receptors and their ligands are

listed in Table 1.

TABLE 1 – Platelet membrane receptors and their ligands

Platelet Receptor Ligands

GP IIb/IIIa, integrin αIIbβ3 Fibrinogen, vWF, Fibronectin

GP Ib-V-IX vWF

GP Ia/IIa, α2β1 Collagen

Protease activatable receptor (PAR) Thrombin

α- adrenergic sites Epinephrine

GP IV, GP VI Collagen

P2Y1 and P2Y12 ADP

4

Figure 1 – Platelet receptors and ligand interactions (Image taken from Rivera et al

2009).

The platelet membrane receptors bind extracellular factors in response to platelet

activation by different agonists, resulting in platelet adhesion and aggregation. PAR-1 and

PAR-4 in the platelet membrane bind thrombin and mediate adhesion. Collagen can bind

to either GP VI or integrin α2β1. Von Willebrand Factor (vWF), epinephrine and

thromboxane A2 (TxA2) bind to GP Ib/IX/V, α2A and thromboxane receptor (TP)

respectively. The binding of these agonists transmits intracellular signals leading to

elevation of cytosolic Ca2+

, cytoskeletal changes, secretion of agonists such as ADP (that

activates G protein-coupled P2Y1 and P2Y12 receptors), and activation of the integrin

αIIbβ3 that binds vWF or fibrinogen and mediates platelet aggregation

5

a) GP VI – It is a 62kDa platelet–specific type 1 transmembrane glycoprotein of the

immunoglobin superfamily. It is an important collagen receptor of high potency in terms

of initiating platelet activation, aggregation and thrombus formation (Nieswandt et al

2003). It is one of the most important members of immunoglobin (Ig) suprefamily on

platelets. It binds ligands including collagen, collagen related peptide (CRP) and the

snake venom protein, convulxin.

Fc receptor gamma (FcRγ) is required for GP VI expression and is associated with

the GP VI via a salt bridge in the transmembrane domains of GP VI (arginine) and FcRγ

(aspartic acid) (Farndale et al 2004). The signalling pathway involves the FcRγ- chain

and the Src kinases (likely Fyn/Lyn), the adapter protein; linker of activated T cells

(LAT), and leads to the activation of phospholipase C gamma 2 (PLCγ2). GP VI initiates

binding to fibrillar collagen under flow conditions, which then activates integrin α2β1

which binds collagen more tightly. GP VI deficiencies cause only a mild bleeding

tendency, probably because integrin α2β1 is able to minimally initiate collagen binding.

b) GP Ib-IX-V – GP Ib-IX-V consists of four transmembrane glycoproteins, which are

all members of the leucine rich protein family (Berndt et al 1995). It is a complex of

glycoproteins constituting GP Ibα and GP Ibβ linked by a disulfide bond, and non

covalently linked to GP IX and GP V. This receptor is constitutively expressed on the

platelet plasma membrane. GPIbα is the major ligand-binding subunit, with a globular N-

terminal ligand-binding domain elevated from the cell surface by a sialomucin core

(Andrews et al 2003). It also plays a substantial role in platelet interaction with activated

endothelial cells and with leukocytes, through the binding of P-selectin and Mac-1

(αMβ2), respectively. There are around 25,000 copies of the GP Ib-IX complex per platelet

and there are approximately half as many copies of GP V per platelet, which suggests that

GP V forms a 1:2 complex with GP Ib-IX on the platelet surface (Modderman et al

1992).

Under high shear, GP Ib-IX-V complex interacts with vWF, an extracellular

multimeric adhesive glycoprotein associated with subendothelial matrix, and mediates the

initial adhesion of platelets to the subendothelium. vWF undergoes a conformational

6

change when it is bound to matrix or under high shear conditions, which permits its

binding to GP Ib-IX-V complex.

Figure 2 – Platelet signalling through GP VI and GP Ib/IX/V (Image taken from

Rivera et al 2009).

GP VI has a short cytoplasmic tail which binds Fyn and Lyn Src kinases. FcRγ complexes

with GP VI, has an immunoreceptor tyrosine based activation motif (ITAM) which acts

as the signal transducing subunit of the receptor. Collagen binding to the GP VI

phosphorylates ITAM by Src kinases which activate Syk and downstream signalling

pathways. These downstream pathways consist of formation of a signalosome, composed

of various adapter and effector proteins (LAT, SLP-76, Gads), and ultimately activates

PLCγ2. PLC catalyzes the hydrolysis of phosphatidyl inositol 4, 5 bisphosphate (PIP2),

thus leading to release of inositol 1, 4, 5 triphosphate (IP3) and diacylglycerol (DAG) and

hence integrin activation. The cytoplasmic tail of GP Ibα is associated with filamin and

calmodulin, which links it to the relevant signalling proteins including Src related

tyrosine kinase, focal adhesion kinase (FAK), GTPase activating protein and PI3K. When

vWF binds GP 1b/IX/V, activation signals such as cytoplasmic Ca2+

release, ADP release

and αIIbβ3 activation are elicited. The actual mechanisms as to how these signals are

transmitted are not yet clear.

7

c) Integrins – Integrins are a ubiquitously expressed family of transmembrane receptors

which consist of an α and β subunit. Integrins are present on platelet membrane in a low

affinity state until platelets are activated by agonists. Platelet activation transforms

integrins into a high affinity form and they can bind their ligands. Integrin αIIbβ3 receptor

is a dominant receptor, which is present in large copy numbers (40,000 – 80,000 per

platelet) on the platelet surface. It acts as a principle receptor which mediates platelet

aggregation through binding of plasma fibrinogen (Gruner et al 2003). Its absence or

deficiency causes the most common bleeding disorder Glanzmann thrombasthenia.

The second most important integrin receptor on platelet surface is α2β1. It is the

major collagen adhesion receptor and is present at about 2000 – 4000 copies per platelet.

The α2β1 integrin, commonly referred to as GP Ia/IIa, also plays a role in the adhesion of

platelets to collagen and subsequent optimal activation (Sarratt et al 2005).

8

Figure 3 – Integrin activation (Image taken from Nieswandt et al 2009).

Phospholipase C (PLC) is activated after stimulation by the agonists. PLC catalyzes the

hydrolysis of phosphatidyl inositol 4,5 bisphosphate (PIP2) into inositol 1,4,5 triphosphate

(IP3) and diacylglycerol (DAG). IP3 binds and activates IP3 receptors on the endoplasmic

reticulum (ER) membrane resulting in the release of calcium from the ER. Ca2+

concentration inside the cell is further increased by the opening of plasma membrane Ca2+

channel Orail by stromal interaction molecule 1 (STIM1). DAG and Ca2+

activate protein

kinase C (PKC) and diacylglycerol-regulated guanine nucleotide exchange factor I

(CalDAG-GEFI), which further activates and translocates Rap1 to the plasma membrane.

RAP1 effector molecule RIAM interacts with Rap1-GTP and talin-1. This interaction

exposes the integrin binding site of talin-1. The salt bridge between the transmembrane

regions of α and β integrin subunits is disrupted by the binding of talin-1, which results in

a conformational change in the extracellular domains and hence ligand binding. This step

also requires binding of kindlin-3 to the NPXY motif of the integrin β tail.

9

d) ADP receptors – ADP plays a central role in regulating platelet function. It induces

platelet aggregation via the activation of 2 major ADP receptors, P2Y1 and P2Y12. ADP

binds the Gq-protein-linked P2Y1 receptor on platelets, which causes a change in cell

shape, mobilization of calcium, and initiation of reversible aggregation. It also binds the

Gi-linked P2Y12 receptor to amplify aggregation via adenylyl-cyclase-mediated cyclic

AMP production (Communi et al 2000). This receptor is also a main target for many anti-

thrombotic drugs. Coactivation of Gq-coupled P2Y1 and Gi-coupled P2Y12 receptors is

essential for ADP-induced platelet aggregation (Jin et al 1998).

10

Figure 4 – ADP receptors and downstream signalling (Image taken from Kim et al

2011).

ADP activates platelets through Gq coupled P2Y1 receptor and Gi-coupled P2Y12 receptor

and causes a number of downstream intracellular signalling events that contribute to

fibrinogen receptor activation and platelet aggregation. G protein gated inwardly

rectifying potassium channels, PI3K, Akt, ERK, Rap 1b and Src family kinases are all

activated through the P2Y12 receptor. Both Rap1b and Akt are signalling mediators that

contribute to platelet aggregation and are activated in a PI3K-dependent manner. RhoA

protein is activated by P2Y1, which causes cytoskeletal and shape changes in activated

platelets. These receptors are also the target of many anti-thrombotic drugs including

clopidogrel, prasugrel and elinogrel. MRS2179 is P2Y1 antagonist.

11

1.2 Major proteins involved in thrombosis

Blood coagulation cascade involves numerous different proteins eg fibrinogen, collagen,

tissue factor, factor VII, factor VIII, vWF etc. The properties and functions of 3 major

proteins related to the work in this thesis are described below.

a) Fibrinogen – Fibrinogen is a large (340 kDa), complex glycoprotein which is

primarily synthesized by hepatocytes. It consists of three pairs of polypeptide chains, Aα,

Bβ and γ, linked by 29 disulfide bonds. These polypeptide chains are encoded by different

genes located on chromosome 4. It is normally present in blood plasma at a concentration

of about 2.5g/L with a half life of around 100 h. The primary platelet receptor for

fibrinogen binding is integrin αIIbβ3 (Weisel 2005). Fibrinogen is essential for hemostasis

and plays an important role in wound healing, inflammation and other biological

functions.

Fibrinogen is not only necessary for platelet aggregation, which is an initial step in

hemostasis but also in the formation of insoluble fibrin clots in the final stages of the

blood coagulation cascade. Fibrinogen binds to integrin receptor αIIbβ3 on the activated

platelets and act as a bridge to link platelets causing platelet aggregation and hence

thrombus formation. Human fibrinogen contains three integrin binding sites: two

arginine-glycine-aspartic acid (RGD) sequences within the Aα chain and a non RGD

sequence in the γ chain (Weisel 2005).

Fibrinogen also plays a role during inflammation and immune response and

mediates the adhesion and transendothelial migration of leukocytes. The synthesis of

fibrinogen is increased during inflammation. Two leukocyte integrins, αMβ2

(CD11b/CD18, Mac-1) and αXβ2 (CD11c/CD18) are the main fibrinogen receptors

expressed on neutrophils, monocytes, and macrophages. Fibrinogen interacts with

CD11b/CD18 and intercellular adhesion molecule-1 (ICAM-1) on endothelial cells and

acts as a bridging molecule to enhance leukocyte adhesion to endothelial cells (Languino

et al 1993; Simmons et al 1988).

b) Collagen – Collagen is the most abundant protein in the human body and a major

protein of the extracellular matrix. Collagen plays a major role in the hemostatic cascade.

Platelet adhesion and aggregation on collagen is an integrated process that involves

12

various platelet receptors, including GP VI and integrin α2β1. The human genome has

genes for more than 20 forms of collagen. Approximately 9 forms of collagen are

expressed in the vascular wall (Type I, III, IV, V, VI, VIII, XII, XIV), of which types I

and III are the major constituents of the extracellular matrix. Repetitive Gly–X–Y

sequences are the characteristic feature of the primary structure of triple-helical regions of

collagen. Gly– Pro–Hyp (GPO) is the most prevalent sequence, which forms about 10%

of the primary structure of collagen types I and III (Baum et al 1999). For the close

packing of the chains, every third residue must be glycine.

GP Ia/IIa (α2β1) and GPVI are the two major collagen binding receptors present on

platelet membrane. Under normal conditions, collagen is not exposed to flowing blood.

After vascular injury, collagen becomes exposed to the flowing blood; the platelets

adhere to the matrix and form an aggregate. Under high shear rates, platelet adhesion to

collagen requires vWF (Sixma et al 1997).

c) von Willebrand Factor (vWF) – vWF is a multimeric adhesive glycoprotein present

in the plasma, the subendothelial matrix and on the surface of activated endothelial cells.

vWF is stored in storage granules (Weibel-Palade) of endothelial cells and α granules of

platelets and is released upon activation of both types of cells. vWF has binding sites for

platelet GP Iba and GP IIb-IIIa, and for various subendothelial constituents, including

collagen types I, III, and VI. For the initial tethering of flowing platelets at the very high

shear rates found in small arteries and arterioles, the interaction between glycoprotein Ib-

V-IX (GPIb-V-IX) and von vWF immobilized on collagen is very critical (Moroi et al

1999).

1.3 Methods to study platelet function

1. Light transmission aggregometery

Aggregometry has been used as a standard approach to study platelet aggregation in

research. The use of light transmission through platelet suspensions to determine

aggregation was introduced in the early 1960s (Born et al 1962). Shape change and

aggregation can be studied with a platelet aggregometer, which records the transmission

13

of light through a stirred platelet suspension. The agonist (ADP, thrombin, collagen) is

added to the platelet suspension and the dynamic measure of platelet aggregation is

recorded over time at 600nm. This platelet aggregation test can be done by using platelet

rich plasma (PRP) maintained at 37o C. When the agonist is added, the platelets aggregate

and absorb less light and so the transmission increases. The change in optical density can

be plotted to view the aggregation curve. Lumiaggregometry is a modification of light

transmission aggregometry which measures platelet secretion along with platelet

aggregation.

2. Cone and plate viscometers

Fluid mechanical forces can exert profound effects on blood cell function. Over the past

years, shear forces generated by blood flow have been recognized to have a significant

impact on platelet adhesion and thrombus formation. Cone and plate viscometers have

been used for the continuous measure of platelet agglutination generated by shear forces

(Fukuyama et al 1989). The role of shear forces in inducing platelet activation has been

measured by subjecting platelets in suspension to a range of shear rates in a viscometer

(Goto et al 1998, 2002; Ikeda et al 1991).

As shown in Fig. 5, the cone is of a very shallow angle and is in bare contact with

the plate. The platelet suspension is placed between the cone and plate and rotation of the

cone at a calculated rate induces shear in the suspension. The rate of shear as well as the

shear patterns can be controlled by the angle of the cone (α), the speed of rotation (ω), the

viscosity of the medium (µ) and the distance between the cone (h(r)) and the plate. The

platelets can be exposed to different range of shear stress and the sample volume required

is around 500µL – 2mL.

A major advantage of a cone and plate viscometer is the constant shear rate

throughout the entire sample. It requires very low volumes of blood/platelet suspensions

and can be used to study both laminar and turbulent flows. It allows measurement of

relatively high shear rates, requires small sample volumes and is easy to clean. The main

disadvantage of the cone and plate viscometer is that it has geometric disparity from the

vessels in the cardiovascular system. Also, it cannot be used to study platelet function in

real time.

14

Figure 5 – Cone and plate viscometer.

3. Perfusion chambers

Wall shear has been identified as an important parameter governing the growth of platelet

thrombi. The platelets experience shear stresses in the range of 1 – 60 dyne/cm2 in the

venous and arterial circulation. The perfusion chambers are used to study the influence of

laminar blood flow at various shear rates on thrombus formation.

A perfusion device consists of a perfusion chamber with a glass coverslip, a

pump, vials containing blood/platelet samples and tubing to connect the chamber and the

pump. The matrix proteins (collagen, fibrinogen) are coated on the glass coverslips. The

main advantage of these perfusion chambers is that they allow studies at various

pathophysiological shear rates and also, coating the chamber surfaces with matrix

proteins is a very simple procedure. These perfusion chambers also allow us to study the

dynamics of platelet aggregation in real time. The limitation of these chambers is that

blood flow is pulsatile and blood vessels can dilate, whereas in these flow chambers the

blood flow is constant and walls are rigid.

Parallel plate flow chambers are the most commonly used perfusion chambers to

mimic flow conditions occurring in vivo and study platelet function in vitro. The role of

various platelet membrane receptors, adhesive proteins at the vessel wall, plasma

proteins, thrombin formation and shear rate on platelet adhesion and aggregate formation

can be investigated using these perfusion chambers. Various studies have used these

15

parallel plate flow chambers to determine the interaction of platelets with adhesive

surfaces (collagen, fibrinogen, vWF) at various shear rates (Savage et al 1996; Loncar et

al 2006). Tubular flow chambers which retain the cylindrical shape of the vasculature

have been used to study the growth and stability of platelet aggregates and also to study

the flow mechanisms for depositing platelets on various surfaces (Badimon et al 1987).

Flow chambers with built in eccentric cosine shaped stenoses in the blood flow channel

have been used to study the effects of fluid dynamic factors on thrombus formation at

arterial stenotic lesions (Barstad et al 1994). Apart from the conventional flow chambers,

microfluidic devices are also used, which reduce the blood volumes required for platelet

studies (Gutierrez et al 2008).

16

Figure 6 – Perfusion Chamber (Image taken from www.dagan.com)

17

2. Shear Stress

In normal physiological conditions, various mechanical forces act on blood vessels and

the endothelial cells lining them. Two types of superficial stresses develop at the vessel

walls due to blood flow – circumferential stress due to variation of pulse pressure inside

blood vessels and shear stress due to blood flow. These forces consist of pressure acting

perpendicular to the vessel wall, cyclic strain, and shear stress acting parallel to the wall,

creating a frictional shear force on the surface of the endothelium. Shear stress is

measured in dynes/cm2. For Newtonian fluids flowing upon a planar surface, shear stress

is calculated as

τ = μ du/dy

where τ is shear stress, μ is kinetic viscosity, u is fluid velocity, y is distance from the

surface and du/dy is the velocity gradient (Shames et al 2003).

The arteries and veins are exposed to different levels of shear stress, which may

produce alterations in the structure, exposure, or clustering of externally oriented

molecules in cell membranes. Normal time-average levels of fluid shear stress in the

venous circulation are approximately 1 – 6 dyne/cm2, and in the arterial circulation

approximately 5 – 60 dyne/cm2. In contrast, higher shear stress values can be observed in

arteries with strong curvatures such as aortic arches, arterioles and vasculature partially

obstructed by atherosclerosis (McDonald et al 1974).

2.1 Role of shear stress in platelet adhesion and aggregation

Shear stress has an important role in maintaining vascular homeostasis. Various studies

have focused on the physiological effects of shear stress. Blood rheology is one of the key

factors regulating the dynamics of thrombus development. The mechanisms of platelet

deposition and thrombus growth are to a large extent determined by the alterations in the

local hemodynamic environment (Mustard et al 1966). In healthy arteries, blood flow is

laminar and platelets are exposed to uniform hemodynamic forces during hemostatic plug

formation. At the site of vascular injury, shear forces generated by the flowing blood play

an important role in platelet adhesion mechanisms. These mechanical forces not only

transport platelets to the vessel wall but also dictate the role of various receptors in

18

mediating platelet adhesion. Activation of platelets by pathologically high shear stress can

lead to arterial thrombotic disease.

High fluid shear may trigger platelet aggregation (Kroll et al 1996; Andrews et al

1997). This condition is known as shear induced platelet aggregation (SIPA) and is

known to play an important role in the pathogenesis of various diseases including

atherosclerosis and acute myocardial infarction. Rapid and dramatic changes in blood

flow may activate passing blood platelets. Various studies on effects of shear rates have

shown that increasing shear stress leads to increased deposition of platelets onto

thrombogenic surfaces and increased rates of thrombus growth (Turitto et al 1979; Ikeda

et al 1991; Tsuji et al 1999; Ruggeri et al 2006). A recent study has demonstrated that at

pathological shear rates (> 10,000s-1

), large rolling aggregates can develop independently

of integrin αIIbβ3 and platelet activation (Ruggeri et al 2006).

Platelet aggregation is a consequence of the bridging of platelet surface integrin

αIIbβ3, by fibrinogen. Although the receptor complexes in resting or unactivated platelets

do not bind the bridging ligand, high shear forces are thought to induce conformational

changes in the GpIb/IX/V complex or vWF, and consequent platelet aggregation via the

bridging molecule vWF (Ikeda et al 1991; Kroll et al 1996). vWF binding to αIIbβ3is

minimal, but when high shear stresses are applied to platelets, vWF binds to αIIbβ3 as well

as to the GP Ib/IX/V complex, and this binding contributes substantially to direct shear-

induced platelet aggregate formation (Goto et al 1995).

In small arteries and arterioles where shear rate is very high, initial platelet

adhesion depends on the binding of GP Ibα to immobilized vWF. This interaction is

crucial for the initial tethering of flowing platelets. This complex continues to recruit

platelets, thereby increasing the shear rate due to growing thrombi, however this binding

is not sufficient to make stable platelet aggregates and the platelets continue to be

translocated in the direction of blood flow. It can keep platelets in contact with the surface

and with each other only for a short time. High shear stress induces binding of platelet

GP-IX-V to plasma von Willebrand factor (vWF) which initiates platelet aggregation

(Savage et al 1998). This molecular mechanism initiates various signalling pathways

which lead to increased intracellular calcium and αIIbβ3 integin receptor activation. This

leads to thrombus formation which can block blood supply to the heart and brain causing

19

heart attack and stroke (Kroll et al 1996; Gawaz et al 2004). Activated platelets also

interact with leukocytes circulating in the blood flow and mediate platelet-leukocyte-

endothelial cell adhesion. In atherothrombosis, platelets promote the interaction of

inflammatory leukocytes with the vessel wall, which initiates formation of atherosclerotic

plaques (Gawaz et al 2004; Massberg et al 2002).

2.2 Endothelial cell responses to shear stress

Endothelial cells constitute the inner lining of blood vessels and constantly experience

fluid shear stress, the tangential component of hemodynamic stresses. When shear stress

is applied on the luminal surface of endothelial cells, the mechanical-chemical signaling

can be transmitted throughout the cell and to cell– extracellular matrix (ECM) adhesions

on the luminal surface of endothelial cells. Endothelial cell surfaces are equipped with

various mechanoreceptors which convert physical stresses into biochemical signals.

Endothelial cells sense shear stress and respond by modifying gene expression,

intracellular signalling, protein expression and other cell functions. Under normal

conditions, the blood flow is unidirectional and laminar, whereas low and disturbed blood

flow promotes development of atherosclerotic plaques (Davies et al 1995).

Numerous studies have been done in vitro and in vivo to demonstrate the effects of

shear stress on the morphology and cellular behaviour of endothelial cells. Sustained

shear stress results in reorientation of the actin cytoskeleton, microtubules, and

intermediate filaments in the direction of flow (Malek et al 1996; Girard et al 1995).

Some of the early responses of endothelial cells to shear stress such as the activation of

Ca2+

and K+ transmembrane channels (Yoshikawa et al 1997) and platelet endothelial cell

adhesion molecule 1 (PECAM-1) phosphorylation (Osawa et al 1997) can be detected

within a few seconds after the onset of flow. Shear stress also leads to the activation and

phosphorylation of various signalling molecules e.g. MAP kinases (Tseng et al 1995),

focal adhesion kinase, jun C terminal kinase (Li et al 1997) and protein kinase C (Traub

et al 1997). Laminar shear stress also enhances endothelial cell migration in wound

healing (Li et al 2002; Hsu et al 2001; Albuquerque et al 2000). Physiological shear

stress decreases the rate of apoptosis from growth factor depletion, tumor necrosis factor

α or hydrogen peroxide exposure (Levesque et al 1990; Chiu et al 1998) via activation of

20

Akt and attenuated caspase mediated killing (Dimmeler et al 1996).

Prolonged shear stress causes distinct structural and morphological changes in

endothelial cells and also affects the overall vascular tone through the regulation of

various vasoconstrictors and vasodilators. Shear stress is essential to maintain endothelial

integrity and cardiovascular health. It has been shown that fluid shear stress plays an

important role in blood vessel formation and maintenance although the mechanisms are

not fully understood yet.

2.3 Shear stress sensors

The ability of endothelial cells to respond to shear stress indicates that they can sense

shear stress as a signal. Various studies have been done to understand the mechanisms of

signal mechanotransduction in shear stress. Shear stress is detected by various receptors,

tranducers and sensor proteins on the cell surface and multiple pathways are involved in

the shear stress signal transduction including G proteins, tyrosine kinase receptors,

caveolae and ion channels.

21

Figure 7 – Mechanotransduction of endothelial shear stress (Image taken from

Chatzizisis et al 2007).

Shear stress is sensed by various mechanoreceptors including integrins, caveolae, ion

channels, G coupled proteins, tyrosine kinase receptors and proteoglycans. Integrins are

activated when shear stress signals are transmitted through the cytoskeleton to the basal

endothelial surface. Various other proteins and protein complexes including adaptor

proteins (Grb2, Crk), non receptor tyrosine kinases (FAK, c-Src, Shc, paxillin, p 130CAS

)

and guanine nucleotide exchange factors (Sos and C3G), are phosphorylated and

activated by the integrins. This activates the Ras family GTPase, which triggers

downstream cascades of serine kinases, ultimately activating mitogen activated protein

kinases (MAPKs). Shear stress signals transmitted through the cytoskeleton to the

junctional or luminal endothelial surface activates protein kinase C (PKC), Rho family

small GTPases which mediate cytoskeletal remodelling and phosphoinositide 3 kinase

(PI3K) - Akt cascade. All of these signalling pathways lead to phosphorylation of various

transcription factors, which bind to shear stress responsive elements (SSPE) at promoters

of mechanosensitive genes, thereby inducing or suppressing their expression

22

2.4 Shear stress, endothelial dysfunction and atherosclerosis

Atherosclerosis is a chronic and inflammatory disease of conduit arteries and is the

leading cause of death in developed countries. It is associated with several other well

defined risk factors including hypertension, hyperlipidemia and diabetes mellitus. The

atherosclerotic lesions form at specific regions of the arterial tree, such as in the

vicinity

of branch points, the outer wall of bifurcations, and the inner wall of curvatures, where

disturbed flow occurs. It has been widely shown that atherosclerosis preferentially

develops in vascular regions with low shear (Malek et al 1999; Nerem et al 1993). On the

other hand, the vessel regions which are exposed to laminar, steady and high shear flow

remains disease free.

Sterol regulatory element binding proteins (SREBPs) are also activated by low

shear stress. These endoplasmic reticulum-bound transcription factors upregulate the

expression of genes encoding the LDL receptor, cholesterol synthase, and fatty acid

synthase (Liu et al 2002). SREBPs also increase the permeability of the endothelial

surface to LDL (Traub et al 1998) and production of reactive oxygen species (ROS). The

circulatory inflammatory cells (monocytes, T lymphocytes and mast cells) are recruited

into the tunica intima, the innermost layer of the arteries, to scavenge oxidized LDL.

NFκB is activated which upregulates genes encoding vascular cell adhesion molecule-1

(VCAM-1), intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant

protein (MCP)-1; and pro-inflammatory cytokines, such as tumor necrosis factor α (TNF

α) and interleukin (IL)-1. These adhesion molecules mediate rolling and adhesion of

leukocytes on endothelial cells. MCP-1 promotes recruitment of leukocytes into the

intima. After leukocyte infiltration into endothelial cells, they differentiate into

macrophages, which sustain inflammation and hence promote atherosclerosis.

Shear stress is also associated with endothelial proliferation. Endothelial cell

proliferation increases by 18 times within 48 hours of shear stress reduction (Mondy et al

1997). Decrease in shear stress also causes vascular smooth muscle cell proliferation,

differentiation and migration into the intima, endothelial cell loss and decrease in actin

stress fibres (Walpola et al 1993, 1995).

23

Figure 8 – Effects of disturbed shear rates on endothelial cell function (Image taken

from Malek et al 1999).

Hemodynamic shear stress is very important for maintaining endothelial integrity and

function. Arterial-level shear stress (>15 dyne/cm2) induces endothelial quiescence by

decreasing proliferation and apoptosis. It also upregulates the expression of

atheroprotective genes including eNOS. However, low shear stress (<4 dyne/cm2), which

is prevalent at atherosclerosis-prone sites, stimulates an atherogenic phenotype by

decreasing antioxidants production and atheroprotective genes. Increased expression of

VCAM and MCP-1 activates the monocytes, enhancing the progression of atherogenesis.

24

2.5 Parallel plate flow chambers

To study the dynamic response of vascular endothelial cells to controlled levels of fluid

shear stress, various types of in vitro systems that allow cultured endothelial cells to be

exposed to well-defined flow conditions have been developed. These systems include a

cone-plate apparatus (Dewey et al 1981), orbital shakers (Dardik et al 2005), capillary

flow tubes (Olesen et al 1988; Jacobs et al 1995), and parallel-plate flow chambers

(PPFC) (Ruel et al 1995; Chiu et al 1998).

Among in vitro systems employed to study the effects of flow conditions on

endothelial cells, PPFC have been the most commonly used for flow stimulation of

endothelial cells (Brown et al 2000). PPFC are generally used to mimic shear flow on

cultured endothelial cells. A typical PPFC consists of a silicon gasket, a polycarbonate

distributor, which has inlet and outlet ports, and a glass coverslip on which endothelial

cells are grown. The endothelial monolayer is subjected to fluid flow by creating a

pressure gradient along the chamber. The design of PPFCs is very simple and they are

very easy to operate. The main advantage of PPFC is that it can be used to produce shear

rates ranging from 0.01 – 60 dyne/cm2 and studies can be visualized in real time.

Studies using the PPFC have provided insight into the effects of shear on

endothelial cell alignment and elongation, aided investigators in fine tuning the

application and adjustment of shear and helped lay the groundwork for future devices.

For example, endothelial cells have been subjected to steady and pulsatile shear stress to

measure and correlate their production of prostacyclin (Hanada et al 2000; Grabowski et

al 1985), determine their orientation with respect to flow direction and reveal that higher

shear stress results in a higher degree of cell elongation

(Levesque et al 1985).

Mechanotransduction in endothelial cells has been studied using many in vitro and in vivo

approaches.

PPFCs have been used to study the effects of shear stress on the apoptosis of

HUVECs induced by lipopolysaccharide (LPS) (Zeng et al 2005) and expression of

proto-oncogenes, c-fos and c-myc in HUVECs (Li et al 2002). The effects of fluid shear

stress on MCP-1 induction in endothelial cells have also been investigated using PPFCs

(Yu et al 2002). PPFCs have also been used to study the mechanisms underlying

proliferation, adhesion and metastasis of cancer cells (Zhang et al 2003).

25

3. Thymosin β 4

Thymosins are a group of peptides which were first isolated from calf thymus. These are

divided into 3 different groups based on their isoelectric point: α thymosin (pI < 5), β

thymosin (5 < pI < 7) and γ thymosin (pI > 7). β thymosins are a family of structurally

related proteins, which have highly conserved amino acid sequences among different

species. Thymosin β 10 and thymosin β 15 are expressed in breast, thyroid and prostate

metastatic tumors and are thought to be the prognostic markers of these cancers (Bao et al

1996; Santelli et al 1999). There are 16 members of the β thymosin family and thymosin

β 4 (Tβ4) is the most studied of all the members of the family.

Tβ4 is a highly conserved peptide, which was first isolated from thymus tissue in

1981 (Low et al 1981; Yu et al 1994). It has 43 amino acids with a molecular weight of

4.9 kDa. It is found in concentrations of 10 nM – 600 M in different tissues and cell

types (Hannappel et al 1985, 1987). The concentration of free Tβ4 in blood is about 10–

200 nM (plasma and serum). It is regarded as a major intracellular G actin sequestering

peptide in mammalian cells. It forms a 1:1 complex with G actin and inhibits its salt

induced polymerization to F actin (Hannappel et al 1993; Huff et al 2001). Tβ4 is also

present inside the cell and extracellular fluid.

3.1 Structure and properties

Tβ4 has a dynamic and flexible conformation. Three dimensional structure

determinations by NMR-techniques have demonstrated that Tβ4 is unstructured in

aqueous solutions. However, when fluorinated alcohols are added to the aqueous solution

a defined 3D-structure can be obtained (Zarbock et al 1990). The data obtained under

these conditions indicated that Tβ4 is a rather extended molecule of almost 5 nm in

length. Both the N- and C-terminal conserved sequence stretches ranging from residues 5

to 15 and 30 to 40, respectively, form short α-helices linked by a flexible loop.

Proteins which lack a definite secondary structure in aqueous solutions attain a

defined tertiary structure on binding their target proteins and undergo a number of

structural intermediate states after forming the initial collision complex (Sugase et al

2007). Due to their ability to attain different conformations, such proteins can also

interact with a number of different proteins. β thymosins might also behave in a similar

26

way. In other words, their binding to actin may induce a stable three-dimensional

structure with defined secondary structural elements. The ability to adopt different

conformations might explain the promiscuous protein interactions and multiple

extracellular functions of β thymosins.

3.2 Biological functions

Various studies have shown diverse biological roles of Tβ4. It promotes wound repair,

angiogenesis, cell migration, tissue protection, regeneration in the skin, eyes and heart,

and prevents apoptosis and inflammation.

3.2.1 Tβ4 promotes cell migration and adhesion

The regulation of polymerization and depolymerization of actin subunits is a key

mechanism by which Tβ4 enables cells to migrate (Malinda et al 1997; Grant et al 1995).

It also upregulates the gene expression of laminin -5, a subepithelial basement membrane

protein believed to be one of the best ligands for keratinocyte adhesion and migration,

and influences cell migration (Sosne et al 2004). Tβ4 also activates Akt, which plays a

very potent role in cell growth, survival and motility (Bock-Marquette et al 2004). During

the morphological differentiation of endothelial cells into capillary-like tubes, there is a

five fold increase in Tβ4 mRNA level. When the endothelial cells are transfected with

Tβ4, there is an increased rate of attachment and spreading on matrix components and an

accelerated rate of tube formation on Matrigel (Grant et al 1995). Tβ4 also stimulates the

migration of HUVECs (Malinda et al 1997) and induces matrix metalloproteinase 2 in

vitro and in vivo (Malinda et al 1999). Another study indicated that local production of

Tβ4 is increased during muscle injury and it promotes myoblast migration, hence

facilitating skeletal muscle regeneration (Tokura et al 2011).

3.2.2 Tβ4 prevents apoptosis and promotes cell survival, angiogenesis and cell

differentiation

Tβ4 has been shown to reduce apoptosis and promote induction of anti apoptotic genes. A

previous study on the pro-apoptotic effects of ethanol on corneal epithelial cells has

27

shown that Tβ4 decreases cytochrome c release from the mitochondria and caspase

activation. It also increases the expression of anti-apoptotic protein, bcl-2 (Sosne et al

2004). In cardiomyocytes, it activates the phosphoinositide/Akt cell survival signalling

pathway and inhibits endothelial apoptosis (Hinkel et al 2008).

Tβ4 is known to be an important angiogenic molecule that promotes angiogenesis

by differentiation and directional migration of endothelial cells (Malinda et al 1997;

Grant et al 1999). It promotes coronary vessel development and collateral growth during

embryonic development and also stimulates epicardial vascular progenitors which can

later differentiate into endothelial and smooth muscle cells (Smart et al 2007). Tβ4 has

been shown to induce dermal repair and hair growth by promoting stem cell migration

and differentiation into keratinocytes and hair follicles (Philip et al 2004).

3.2.3 Role of Tβ4 in thrombosis

The concentration of Tβ4 is very high in white blood cells and in plasma is very low,

however during blood clotting, Tβ4 concentration in the serum increases substantially. In

1987, it was shown that Tβ4 is present in human blood platelets in very high

concentrations (Hannappel et al 1987). In resting platelets, the Tβ4 concentration is ~

560µM, of which ~ 280µM is in complex with G actin and ~280µM is free (Nachmias et

al 1993).

During blood coagulation or ADP induced aggregation of platelets Tβ4 is

liberated from platelets and partially cross-linked to fibrin by a transglutaminase, factor

XIIIa (Huff et al 2002). Tβ4 crosslinking to the fibrin occurs in a time and Ca2+

dependent manner. Factor XIIIa incorporates Tβ4 preferentially into the fibrin αC-

domains, a domain often used to attach biologically active peptides to fibrin

(Makogonenko et al 2004). The covalent cross-linking of Tβ4 to the fibrin clot might

represent a mechanism to guarantee a high local concentration of the peptide at the site of

injury probably supporting subsequent wound healing. This could be a potential

molecular mechanism to bring Tβ4 near the site of vascular injury and leads to clotting

and wound repair by Tβ4.

28

Figure 9 – Tβ4 and blood coagulation (Image taken from Huff et al 2002).

When the platelets are stimulated with ADP, various cytoskeletal changes take place. The

platelets change shape and there is secretion of ADP, Factor V, Tβ4, serotonin,

thromboxane A2 and growth factors. The platelets aggregate and form a platelet plug,

which must be stabilized by a fibrin clot to ensure wound closure. The fibrin monomers

attach to the platelet plug and are further cross linked by factor XIII to form an insoluble

clot. Tβ4 released from the platelets is also fixed to the fibrin clot by factor XIII

29

3.2.4 Role of Tβ4 in inflammation and corneal wound healing and repair

Previous studies have shown that Tβ4 decreases inflammation and promotes corneal

wound healing. It decreases matrix metalloproteinases and proinflammatory cytokines

and chemokines. In mouse models, topical application of Tβ4 after alkali injury

downregulates the expression of the potent chemoattractants, macrophage inflamatory

protein 2 (MIP 2) and keratinocyte chemoattractant (KC) in the cornea (Sosne et al 2002).

It also accelerates epithelial cell migration and re-epithelialization after alkaline and

alcohol injuries and scrape wounding in a dose-dependent manner in rat corneal wound

models (Sosne et al 2002, 2005). Tβ4 has been shown to protect corneal endothelial cells

from apoptosis and oxidative stress induced by low dose ultraviolet B (UVB) exposure

(Ho et al 2010). It protects human corneal epithelial cells against Fas ligand (Fas-L) and

H2O2 induced damage. Internalization of Tβ4 is critical for its cytoprotective effect (Ho et

al 2007).

Tβ4 is hypothesized to be an anti-inflammatory agent. It interferes with the NFκB

signalling pathway, which is activated by the potent pro-inflammatory cytokine TNFα.

Tβ4 pre treatment reduces nuclear NFκB protein levels, NFκB activity, and p65 subunit

phosphorylation, and nuclear translocation in corneal epithelial cells stimulated with TNF

α (Sosne et al 2007). No receptors have been identified for Tβ4 thus far. Future

investigation of putative Tβ4 receptors would provide critical information for

understanding how extracellular Tβ4 exerts its biological activities in cells.

30

4. Retinal Pigment Epithelium

Retinal pigment epithelium (RPE) is a monolayer of pigmented cuboidal cells present

between the retinal photoreceptors and choriocapillaris. It is separated from the

choriocapillaris, which supplies blood flow to the RPE, and the outer one-third of the

retina (including the photoreceptors), by Bruch’s membrane. The human RPE

incorporates approximately 3.5 million epithelial cells arranged in a regular hexagonal

pattern. RPE are bound together by junctional complexes with prominent tight junctions.

These junctions divide the cells in an apical half that faces the retina and a basal half that

faces the choroid (Hudspeth et al 1973). The only anatomical contact between the

photoreceptors and RPE is the interphotoreceptor matrix. Under pathological conditions

such as retinal detachment, fluid accumulates in the subretinal space and photoreceptors

are separated from the RPE. This causes a loss of photoreceptor function.

The embryonic development and differentiation of RPE and photoreceptors is

interrelated. Both photoreceptors and choriocapillaries depend on the RPE for their

survival. RPE is crucial for the development of the retina. It secrets various growth

factors required for photoreceptor differentiation and survival. It has been shown in an

animal model of retinal dystrophy, that RPE secretes basic fibroblast growth factor

(bFGF), which promotes the survival of photoreceptors (Faktorovich et al 1990).

Neuronal retina and photoreceptors are affected the most by RPE loss.

31

Figure 10 – Retinal pigment epithelium. (Image taken from www.dev.ellex.com)

4.1 RPE Functions

RPE is involved in a variety of functions which includes phagocytosis of shed outer

segments, transport of vitamin A to the photoreceptors and maintenance of normal

physiology of the choriocapillaries (Bok et al 1993). RPE transports electrolytes and

water from the subretinal space to the choroid, also transports glucose and other nutrients

from the blood to the photoreceptors.

a) Light absorption – RPE increases optical quality by helping in absorption of the

scattered light. RPE pigmentation is very critical to maintain visual function. It contains a

complex composition of various pigments (melanin, lipofuscin) which absorb different

wavelengths of light. Melanin is the main pigment of RPE present within cytoplasmic

granules called melanosomes. It absorbs stray light and minimizes light scattering within

the eye. The retina has a direct and frequent exposure to light which causes the photo-

32

oxidation of lipids, which can be extremely toxic to retinal cells (Girotti et al 2004). The

retina also generates large amounts of reactive oxygen species (ROS) due to its high

oxygen consumption. RPE counterbalances the high oxidative stress present in the retinal

cells by maintaining large amounts of enzymes like superoxide dismutase (SOD) and

catalase (Frank et al 1999; Tate et al 1995) and non enzymatic antioxidants like

ascorbate, lutein and zeaxanthin (Newsome et al 1994; Beatty et al 2000), which act as a

defence system against the oxidative stress.

b) Transport nutrients and ions – RPE transports nutrients like glucose, retinol and

fatty acids from blood to the photoreceptors. It has large numbers of glucose transporters

GLUT 1 and GLUT 3 in its apical and basolateral membranes (Ban et al 2000). With the

help of these glucose transporters, RPE transfers glucose from the blood to the

photoreceptors. GLUT1 induces glucose transport in response to mitogens and hence

adapts glucose transport according to the metabolic needs of the retina. During the visual

cycle, the bulk of retinal is exchanged between RPE and the photoreceptors. The RPE

takes all-trans retinol from the photoreceptors, oxidizes and isomerizes it to 11-cis retinal

and redelivers it to the photoreceptors (Baehr et al 2003). Docosahexaenoic acid (DHA)

is an essential omega 3 fatty acid which is required as a structural element of

photoreceptor and neuron membranes, however, DHA cannot be synthesized by the

neural tissues and is hence transported by RPE from blood to the photoreceptors

(Anderson et al 1992).

Due to the high metabolic turnover in the photoreceptors, a large amount of water

is produced in the retina. Also, there is movement of water from the vitreous body to the

retina due to intraocular pressure (Marmor et al 1990; Hamann et al 2002). Hence, there

is a constant need for removal of water from the retina. RPE transports water and ions

from the subretinal space to the blood (Hughes et al 1998). It also eliminates metabolic

end products of the photoreceptors. Photorecpetor outer segments produce lactic acid and

its subretinal concentration is estimated to be around 19 mM (Adler et al 1992; Hsu et al

1994). RPE removes lactic acid from the subretinal space through lactate-H+

cotransporter MCT1 (Lin et al 1994; Philip et al 1998) and Na+ dependent transporter for

33

organic acids (Kenyon et al 1994). The Na+-K

+-ATPase, which is located in the apical

membrane, provides the energy for transepithelial transport (Marmorstein et al 2001).

c) Phagocytosis – When the photoreceptors are exposed to high intensities of light, there

is an increase in concentration of light induced toxic substances like photo oxidative

radicals and photo damaged proteins and lipids, in the photoreceptors (Beatty et al 2000).

To maintain the excitability of the photoreceptors, the photoreceptor outer segments

undergo a constant renewal process (Young et al 1969; Nguyen-Legros et al 2000). The

highest concentration of these toxic substances is present in the tips of the photoreceptor

outer segments. Hence, these tips are shed from the photoreceptors and new tips are

formed from the base of outer segments, at the cilium. The shedding of tips and the

formation of new tips is very well coordinated, so that the length of photoreceptor outer

segments is maintained. The shed tips are phagocytosed and digested by RPE. Retinal and

docosahexaenoic acid are transported back to the photoreceptors to rebuild the outer

segments (Bok et al 1993; Bibb et al 1974).

d) Secretion – RPE secretes a large number of growth factors which are essential for the

maintenance of the structural integrity of the retina. It secretes fibroblast growth factors

(FGF-1, FGF-2 and FGF-5), insulin like growth factor-1 (IGF-1), VEGF, pigment

epithelium derived factor (PEDF), platelet derived growth factor (PDGF) and lens

epithelium derived growth factor (LEDGF).

Although VEGF is secreted in very low quantities by RPE, it plays an important

role in maintaining intact endothelium of the choriocapillaries (Adamis et al 1993; Burns

et al 1992). A neuroprotective factor PEDF secreted by RPE protects neurons against

glutamate and hypoxia induced apoptosis (Cao et al 2001). It stabilizes the endothelium

of choriocapillaries (King et al 2000) and plays an important role in the embryonic

development of the eye (Behling et al 2002). Another peptide secreted by RPE is

somatostatin, which plays an important role in retinal homeostasis. Somatostatin acts as a

neuromodulator through different pathways including intracellular calcium signalling

(Johnson et al 2001) and glutamate release from the photoreceptors (Akopian et al 2000).

34

e) Retinoid cycle – One of the most important functions of RPE is its role in the visual or

retinoid cycle, which involves repeated movement of retinoid and its derivatives between

photoreceptors and RPE (Bok et al 1993). The light is absorbed by rhodopsin, which is

composed of the G-coupled receptor protein opsin and chromophore 11-cis –retinal. After

light absorption, 11-cis-retinal is converted into all-trans retinal. The photoreceptors lack

cis-trans isomerase function for retinal and are unable to regenerate 11-cis-retinal from

all-trans-retinal. The main purpose of the visual cycle is to regenerate 11-cis retinal,

which acts as a chromophore for the visual pigments of outer segments of photoreceptors.

All-trans-retinal is reduced to all-trans-retinol and is transported to RPE. The

isomerisation is the first step in phototransduction and takes place in RPE. In RPE, retinol

is oxidized and reisomerized to 11-cis-retinal by the enzyme retinal pigment epithelium-

specific proten (RPE65) and is delivered back to the photoreceptors (Hargrave et al 2001;

Pang et al 2006; Bernstein et al 1987). Interphotoreceptor retinoid binding protein (IRBP)

mediates the transport of retinoids between RPE and photoreceptors. IRBP is a large

glycoprotein present in the RPE endosomes and interphotoreceptor matrix (IPM)

(Cunningham et al 2003; Wu et al 2007; Gonzalez-Fernandez et al 2008). IRBP

solubilises retinal and retinol and mediates the direction of transport of these compounds

(Okajima et al 1989; Pepperberg et al 1991).

4.2 RPE ageing and diseases

A variety of structural and biochemical changes occur in RPE with increasing age. There

is increase in atrophy, hyperpigmentation and loss in cell shape. With advancing age,

there is also a decrease in the concentration of RPE cells in the posterior pole. The

melanin content of RPE also decreases with age (Salvi et al 2006). Senescent RPE

accumulate metabolic debris from remnants of incomplete degradation of phagocytized

rod and cone membranes (Ciulla et al 2001). Ingestion of outer segments of

photoreceptors puts a heavy phagocytic burden on RPE, which results in the

accumulation of lipofuscin between RPE and Bruch’s membrane. Lipofuscin is an

undegradable byproduct of outer segment photoreceptor metabolism which increases in

RPE over time. The accumulation of lipofuscin is involved in the pathogenesis of various

35

retinal diseases like age related macular degeneration (AMD) and is one of the major

characteristic features of ageing in RPE. Lipofuscin is continuously exposed to light and

high oxygen tension, which leads to production of reactive oxygen species (ROS) and

causes oxidative damage to mitochondria and mitochondrial DNA. The production of

antioxidants, which RPE produces to counteract oxidative stress, also decreases with age

(Liang et al 2003). Mitochondrial damage affects the cellular and physiological

functioning of RPE. This leads to decrease in energy production and subsequently signals

apoptosis eventually decreasing the number of RPE (Zarbin et al 2004).

Under normal conditions, RPE secretes various growth factors which can protect

photoreceptors from damage. There is constant balance between angiogenic and anti-

angiogenic growth factors within the eye. Imbalance between the secretion of these

growth factors leads to choroidal neovascularisation (Bhutto et al 2006). PEDF and

VEGF are two growth factors secreted by RPE, which cross regulate each other. VEGF

gene expression increases angiogenesis whereas PEDF acts as an antagonist and inhibits

angiogenesis (Tsai et al 2006; Dawson et al 1999). To regulate angiogenesis in the retina,

balance between these two growth factors is very essential (Tong et al 2006). Previous

studies have shown that VEGF is upregulated in RPE and choroid complex in patients

suffering from AMD (Tsai et al 2006). Decreased PEDF levels in the vitreous samples of

AMD patients have also been reported (Holekamp et al 2002).

4.2.1 Age related macular degeneration (AMD) – AMD is the major cause of blindness

in elderly people. The atrophic or dry form of AMD is caused by progressive degradation

of RPE and the photoreceptors. In AMD, vision loss occurs as a result of photoreceptor

damage in the central retina; however the initial pathogenesis involves RPE degeneration

(Zarbin et al 1998). Since RPE is involved in the metabolic and nutritional aspects of the

photoreceptors, RPE dysfunction can cause secondary degeneration of the photoreceptors.

Age-related processes that occur in the retinal pigment epithelium-Bruch’s membrane-

choriocapillaris complex can precede the development of AMD (Liang et al 2003). AMD

is associated with an accumulation of lipofuscin, a pigment formed in tissues with high

levels of oxidative stress. Early AMD is characterized by thickening and loss of normal

36

architecture within Bruch’s membrane, lipofuscin accumulation in the RPE, and drusen

formation beneath the RPE in Bruch’s membrane. The primary clinical characteristic of

late stage dry AMD is the appearance of RPE atrophy. It is characterized by roughly oval

areas of hypopigmentation and is usually the consequence of RPE cell loss. Loss of RPE

cells which provide nutrition to the photoreceptors leads to the gradual degeneration of

nearby photoreceptors, resulting in a progressive visual impairment.

4.2.2 Retinitis pigmentosa – Retinitis pigmentosa is an inherited form of retinal which is

characterized by pigment deposits in the retina. It involves degeneration of photoreceptors

rods and cones and deposits in the retinal pigment epithelium. The accumulation of

metabolic waste products lead to lipofuscin formation which affects the function of the

RPE. The inability of RPE to phagocytose photoreceptor outer segments causes an

autosomal recessive form of retinitis pigmentosa (Edwards et al 1977; Wang et al 2001).

4.2.3 Diabetic retinopathy – Diabetic retinopathy (DR) is one of the leading causes of

blindness in developed countries. DR is characterized by decreased levels of glutathione,

SOD and ascorbic acid (Madsen-Bouterse et al 2008; Silva et al 2009; Minamizono et al

2006). A recent study has shown that there is a decrease in IRBP production in early

diabetic retinopathy (Garcia-Ram´ırez et al 2009). IRBP is required for retinoid transport

between RPE and photoreceptors and is critical for the visual cycle.

Increased production of VEGF, an angiogenic factor, plays an important role in

the pathogenesis of DR. There is a decreased expression of the anti-angiogenic factor,

PEDF, by elevated glucose concentration in cultured human RPE cells (Yao et al 2003).

New therapeutic approaches for DR which involve blocking VEGF and stimulating PEDF

have been proposed. The development of DR is also favoured by the decreased

expression of somatostatin (Carrasco et al 2007). Somatostatin plays an important role in

preventing neovascularisation and fluid accumulation within the retina. These functions

are greatly impaired due to downregulation of somatostatin expression, thus leading to

development of DR (Hern´andez et al 2005; Sim et al 2007).

37

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49

CHAPTER 2

Development of flow device to study effects of shear stress on

endothelial cells and its applications

50

Introduction

Endothelial cells are exposed to various mechanical stimuli such as shear stress,

hydrostatic pressure and cyclic stretch due to vessel deformation. These stimuli alter

endothelial cell morphology, initiate cytoskeletal changes and activate various signalling

pathways. The nature and magnitude of these hemodynamic forces has an important role

in maintaining vascular homeostasis.

Laminar shear stress is one of the most potent endothelial stimulators, which is

very critical for normal vascular functioning. It plays an important role against

inflammatory activation and apoptosis (Tricot et al 2010; Chien et al 2008). Disturbed

shear stress has been implicated in the pathogenesis of various diseases including

atherosclerosis and cardiovascular diseases (Traub et al 1998).

In the past few years, physiological and pathological shear stress has been studied

extensively to understand the development and progression of vascular diseases. To

understand the effects of these hemodynamic forces in vitro, engineered devices are

required that can maintain the endothelial cells in a controlled in vivo like environment.

The fluid flow must be predictable in these devices so that shear stress over endothelial

cells can be properly controlled.

Many in vitro flow systems have been developed to study endothelial cells under

shear stress. These flow devices require a large population of endothelial cells which

needs more chemicals and reagents. Recently, many studies have reported the

development of microfluidic flow chambers. Some of these designs have multiple

microchannels to study different cell types and shear rates at the same time (Young et al

2007). A major drawback of such flow devices is cross contamination between different

channels. Some of these flow devices and microfluidic devices have been custom

laboratory prototypes, while others are commercially available.

In the present study, we describe the construction and testing of a simple and

robust parallel plate flow chamber. The aim of this study was to develop an in vitro model

using a parallel-plate flow chamber to simulate in vivo fluid shear stresses on endothelial

cells exposed to dynamic fluid flow in their physiological environment. A computational

fluid dynamics (CFD) template was developed for simulation and prediction of flow

51

induced shear rates on the endothelium, while an in vitro model was used to investigate

the subsequent cellular biological responses.

The flow chamber was validated by determining changes in the production of

nitric oxide and caveolin-1 expression in bovine endothelial cells exposed to shear stress.

52

Experimental Methods

Cell culture

Bovine aortic endothelial cells (BAECs) were purchased from Coriell Institute for

Medical Research. The cells were cultured in DMEM F-12 media containing 10% fetal

calf serum (FCS, Gibco) and penicillin-streptomycin, at 37°C in a humidified atmosphere

of 95% air and 5% CO2.

Parallel plate flow chamber configuration

The flow apparatus used in this study is a parallel plate flow chamber with a closed loop

system (Figure 1).

CFD simulation and viscosity measurement

In order to use the velocity gradient templates as shear stress templates, the viscosity of

the media at 37ºC was required. A viscometer (Canon Instruments, Model 2085, PA,

USA) and viscosity bath, set to 37ºC, were used. The media was injected into the

viscometer and allowed to reach equilibrium conditions before being suctioned to the top

of the viscometer and then released while the time it takes the fluid to flow through a

defined section was measured. Using equipment specific correlations, the viscosity was

calculated based on the time taken to pass through a defined section. This process was

repeated five times and an average viscosity was calculated and used in the shear stress

calculations. The specific gravity of the media was determined by using a calibrated

hydrometer. The average viscosity was 0.753 cP (0.00753 Pa∙s).

Calculation of shear stress

Shear stress was calculated according to the following formula:

d z

d y

Where μ is the viscosity of the media and dy/dz is the velocity gradient of the flow. From

the templates generated in FLUENT the values of the velocity gradients at various

regions on the plate are known. Since the viscosity of the media has been measured, the

shear stress can be directly calculated and an average shear stress for each region on the

53

template can be defined. The shear stress values used in our investigations ranged from

0.05 to 1.47 Pa, which is within the physiological range for aortas (~0.002 Pa) and veins

(~0.01 Pa).

Shear stress studies

Bovine endothelial cells were grown on coverslips in 35mm cell culture plates. The cells

were exposed to shear stress using a peristaltic pump (Reglo Digital MS 4/6, model ISM

833, Ismatec). PTFE tubing (Scientific Products & Equipment, Canada) was used to link

the pump and the flow chamber. Shear stress experiments were performed for 16 hours in

complete media in 5% CO2 incubator at 37°C.

Measurement of NO production

To determine changes in NO production, endothelial cell monolayers were incubated with

cell culture medium containing 5 M 4,5-diaminofluorescein diacetate (DAF-DA ,

Invitrogen) at 37°C in a 95% air/5% CO2 incubator for 30 min. Cells were then washed

3X with phosphate buffered saline (PBS) and mounted on the stage of an Axiovert 200

inverted fluorescence microscope. DAF-DA fluorescence was monitored using excitation

and emission wavelengths of 485 and 538 nm, respectively.

BAECs were grown under shear stress and static cells were used as control. After

incubation for 24 hours, media containing 100µM of acetylcholine was injected into the

flow chamber. The cells were incubated again and after 1 hr, 200 uL of media was

withdrawn with a Hamilton gastight syringe and directly injected into the purge vessel of

a Sievers Nitric Oxide Analyzer (Model 280i) which contained acetic acid and sodium

iodide.

Immunofluorescence

BAEC cells were grown on coverslips in 35mm cell culture dishes. The cells were grown

under shear stress for 16 hours and static cells were used as control. After incubation, the

cells were washed with PBS and fixed in 3% paraformaldehyde for 10 min. The cells

were then permeabilized with 0.025% Triton X for 10 min. Rabbit polyclonal antibody

against Ser 1177 phospho eNOS (1:200 dilution; Cell Signal) and anti-mouse caveolin-1

54

(1:100) were used as primary antibodies. Alexa Fluor 488 -labelled goat anti-rabbit IgG

and Alexa Fluor 568 conjugated anti-mouse IgG were used as a secondary antibodies

(1:500 dilution; Molecular Probes, Canada). Cells were also stained with either propidium

iodide (PI) or DAPI for the localization of nuclei. Preparations were mounted in

fluoromount G (Southern Biotech) and examined by fluorescence microscopy.

Statistical analysis

Data is expressed as an average of all trials and statistical analysis was done by Student’s

t test. The quantification of the fluorescence intensity was done using Image J software.

55

Results

Flow chamber

The parallel plate flow chamber described herein was designed to minimize solution

volumes, to be operationally robust and precise, simple to set up, and inexpensive. Also

critical to the design was the ability of the chamber to be mounted on a microscope stage

to facilitate visualization experiments. The chamber pictured in Figure 1 is 64 mm in

diameter and 28 mm in height. It was machined out of surplus aluminum and acrylic

stock. The unit consists of three principal elements, an inner acrylic “puck” that forms

the chamber, an interchangeable gasket that creates the channel geometry, and an

aluminum clamping fixture that holds it all together. A special low cost thermoset plastic

has been adapted for use as a chamber gasket. A simple series of die punches provide a

variety of channel geometries. The unit can also accept commercially available gaskets

like those produced by Glycotech (Rockville Maryland). Channel depth is set by the

clamping fixture and the thickness of gasket utilized. The two halves of the clamping

fixture thread together to compress the gasket to provide the chamber seal. To ensure

proper channel depth, two interchangeable locator pins prevent the fixture from being

over tightened. Inlet and outlet ports were machined into the top of the acrylic chamber.

Each is threaded to accept standard commercially available fittings. The ports are

chamfered with a smooth Gaussian shaped profile to minimize flow disturbances during

flow entrance and exit.

56

Assembled Chamber

57

Figure 1 – Schematic diagram of the parallel plate flow chamber.

58

Computational fluid dynamics

To simulate the flow in the chamber and estimate the shear stress for the testing program,

a computational model of the flow channel was developed. The commercial finite volume

solver, Fluent 6.1 was utilized for this study. Free outflow and mass flow inlet boundary

conditions were assumed at the outlet and inlet of the channel respectively in the steady,

laminar simulation. The material properties of the solution were approximated to be that

of water. The inlet/outlet ports and flow channel [19 mm long X 5 mm wide X 0.25 mm

deep] was discretized by tetrahedral elements. Three mesh densities were tested,

consisting of 83,000, 196,000, and 454,000 elements. Values of the flow velocity

assessed at 5 locations across the domain changed by no more than 2% when the mesh

density increased from 196,000 to 454,000. Thus the medium mesh density was chosen

for the test runs.

59

Figure 2 – Velocity gradient template.

A

B

60

Validation of the flow chamber

In the present study, we first examined the morphological changes of endothelial cells

associated with exposure to physiologically relevant shear stress using this flow chamber.

Bovine aortic endothelial cells were either maintained in static condition or exposed to

laminar flow at a shear stress of 5 dyne/cm2 for 16 h. As shown in Figure 3, fluid shear

stress induced dramatic morphological changes such as cell elongation and alignment

with the direction of flow. In contrast, there is no significant cell orientation in cells under

static conditions.

Control 5 dyne/cm2

Figure 3 – Effect of shear stress on morphology of bovine endothelial cells. Bovine

aortic endothelial cells were maintained either under static conditions or under shear

stress (5 dyne/cm2). After incubation for 16 hours, the morphological changes were

visualized using 20X objective of a Zeiss Axiovert 200 microscope.

61

Nitric oxide production and eNOS phosphorylation in response to shear stress

In order to validate whether this flow chamber can be employed for studies in the areas of

biomedical research, the effect of fluid shear stress on nitric oxide production in

endothelial cells was examined. In the present study, nitric oxide production in bovine

aortic endothelial cells was determined by a membrane-permeable fluorescent probe 4, 5-

diaminofluorescein diacetate (DAF-2 DA). A significant upregulation (~ 3.6 fold) of NO

was observed in endothelial cells which were exposed to laminar flow at a shear stress of

5 dyne/cm2 for 16 hours, compared with those maintained in static condition (Fig. 4 A,

B). A qualitative examination of the data revealed that nitric oxide production increased

by 7 fold as compared to the static cells when the endothelial cells were exposed to a

shear rate of 15 dyne/cm2

(Fig. 4 A, B). A significant upregulation (~ 2.5 fold) of

authentic NO (as determined with NO analyzer) was also observed in endothelial cells

which were exposed to laminar flow at a shear stress of 5 dyne/cm2 for 16 hours,

compared with those maintained in static condition (Fig. 4C).

62

Control 5 dyne/cm2 15 dyne/cm

2

0

10

20

30

40

50

60

70

Control 5 dyne/cm2 15 dyne/cm2

DA

F-D

A f

luo

rescen

ce ***

***

A

B

C

63

Control 5 dyne/cm2

Figure 4 – Nitric oxide production and eNOS phosphorylation in response to shear

stress. (A) The bovine endothelial cells grown under static conditions and shear stress

for 16 hours were incubated with 5µM DAF-DA for 30 min. The cells were washed with

PBS and viewed under Zeiss Axiovert fluorescence microscope using 20X objective. (B)

The graph represents average of DAF-DA fluorescence taken from 4 independent

experiments (± SD). *** P < 0.001 (C) Total nitrite was measured in control cells and

cells under shear stress using NO analyzer. The bovine endothelial cells were grown

under shear stress for 16 hours after which the cells were stimulated with acetylcholine

for 1 hour and total nitrite was measured. The data shown is average of 3 independent

experiments (± SD). (D) The cells maintained under static conditions and exposed to

shear stress for 16 hours were fixed with paraformaldehyde and incubated with anti-rabbit

Ser1177 phospho eNOS antibody. Alexa Fluor 488 conjugated anti-rabbit IgG was used

as secondary antibody and the nuclei were stained with PI. The slides were viewed under

oil immersion 40X objective. (E) Average gray values (immunofluorescence intensity) of

phospho eNOS levels in static and shear stress exposed cells. The values (average ± SD)

were calculated from at least 10 different microscopic fields in each slide (n=3). *** P <

0.001.

D

E

64

Change in caveolin-1 expression after shear stress induction

The changes in caveolin-1 expression and distribution were determined by indirect

immunofluorescence. As shown in Fig. 5, the cells exposed to shear stress showed a ~ 2.7

fold increase in caveolin-1 expression as compared to static cells. In static cells, caveolin-

1 is present mostly in the plasma membrane, whereas in cells under shear stress caveolin-

1 is also found in the cytoplasm.

Control 5 dyne/cm2

Figure 5 – Shear stress upregulates caveolin-1 expression in bovine endothelial cells.

(A) BAECs were exposed to shear stress for 16 hours and static cells were used as

controls. Caveolin-1 expression and distribution (red) was determined by indirect

immunofluorescence. The nuclei were stained with DAPI (blue). The images were taken

by 40X oil emersion objective using Zeiss Axiovert 200 microscope. (B) Average gray

values (immunofluorescence intensity) of caveolin-1 levels in cells maintained under

static and shear stress. The values (average ± SD) were calculated from at least 10

different microscopic fields in each slide (n=3). *** P < 0.001.

A

B

65

Discussion

Parallel plate flow chambers have been used for years in the study of hemodynamics and

cellular mechanotransduction (Frangos et al 1985; Levesque et al 1985; Galbraith et al

1998; McCann et al 2005; Bacabac et al 2005). The majority of chambers documented in

the literature were developed for specific research applications and were not mass-

produced for commercial use. The bulk of chambers presently available off the shelf can

cost upwards of $500 for a pump-ready apparatus. They often require the use of a vacuum

line or pump and delicate gaskets to maintain chamber seals and prevent leaks.

This flow device introduced in this study provides a leak proof and contamination

free environment to the cells. It can also be used for real time monitoring of the live cells,

to study the dynamics of endothelial cell response to shear. The materials used for making

this flow device are chemical resistant. Advantages of this system include its simplicity of

use and small number of cells required to run a simulation. The fabrication is convenient,

versatile and economical.

The reorganization of the endothelial cell morphology is one of the earliest

responses in the endothelial cells that are exposed to fluid shear stress. Previous in vivo

and in vitro studies have shown that the endothelial cells elongate and align parallel to the

direction of flow (Azuma et al 2001; Wojciak-Stothard et al 2003). Our results also

showed change in morphology of the endothelial cells, which are in agreement with the

previous reports by other groups (Topper et al 1999; Wang et al 2001; Kadohama et al

2006).

Shear stress is one of the most important physiological stimuli which modulate

many physiological and pathological processes associated with endothelium, such as

inflammatory response, vasodilation and endothelial cell proliferation (Davies et al 1995).

It acts as a potent stimulus for endothelium-dependent NO production and vasodilation

(Kuchan et al 1994).

An important step in the validation process was to determine whether our flow

chamber was able to discern different shear rates. BAECs were exposed to two different

shear rates (5 & 15 dyne/cm2) and changes in NOx production in response to shear stress

were determined. There was a ~ 3.5 fold increase in NOx production at 5 dyne/cm2 and a

66

7 fold increase at 15 dyne/cm2

as compared to the static cells. These results suggest that

this flow chamber can be utilized to attain different shear rates. These findings are also in

agreement with numerous previous studies which demonstrated nitric oxide upregulation

in response of laminar shear stress (Andrews et al 2010; Yang et al 2007).

Previous studies have shown that increase in shear stress increases eNOS activity

by phosphorylation of eNOS at serine 1177 (Corson et al 1996; Dimmeler et al 1999).

The results of this study also illustrated increase in phosphorylation of eNOS at Ser 1177

by 2.3 fold.

Caveolae are small cell surface plasma membrane invaginations, which have

important role in cell signaling and cholesterol homeostasis. Cholesterol binding protein

caveolin-1 is the main marker of caveolae. Caveolin-1 could mediate the transfer of

newly synthesized cholesterol from the ER to the plasma membrane (Smart et al 1996).

Our results are also in agreement with a previous study which has shown that laminar

shear stress increases caveolin-1 expression and it also changes the localization of

caveolin-1 (Sun et al 2002).

Conclusions

In conclusion, we have introduced a parallel plate flow chamber for shear stress studies in

vitro. The data provides robust evidence that this parallel plate flow chamber can be used

as an effective in vitro system to study the effects of fluid shear stress on the structure and

function of endothelial cells. The flow rates can be precisely controlled in this laminar

flow chamber to induce normal stress and shear stress so as to simulate the hemodynamic

environment of human arteries in vivo.

67

References

Andrews AM, Jaron D, Buerk DG, Kirby PL and Barbee KA. Direct, real-time

measurement of shear stress-induced nitric oxide produced from endothelial cells

in vitro. Nitric Oxide 2010; 23(4): 335-342.

Azuma N, Akasaka N, Kito H, Ikeda M, Gahtan V, Sasajima T and Sumpio BE. Role of

p38 MAP kinase in endothelial cell alignment induced by fluid shear stress. Am J

Physiol Heart Circ Physiol 2001; 280(1): H189-197.

Bacabac RG, Smit TH, Cowin SC, Van Loon JJ, Nieuwstadt FT, Heethaar R and Klein-

Nulend J. Dynamic shear stress in parallel-plate flow chambers. J Biomech 2005;

38(1): 159-167.

Chien S. Role of shear stress direction in endothelial mechanotransduction. Mol Cell

Biomech 2008; 5(1): 1-8.

Corson MA, James NL, Latta SE, Nerem RM, Berk BC and Harrison DG.

Phosphorylation of endothelial nitric oxide synthase in response to fluid shear

stress. Circ Res 1996; 79(5): 984-991.Davies PF. Flow-mediated endothelial

mechanotransduction. Physiol Rev, 1995; 75(3): 519-560.

Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R and Zeiher AM. Activation of

nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation.

Nature,1999; 399(6736): 601-605.

Frangos JA, Eskin SJ, McIntire LV and Ives CL. Flow effects on prostacyclin production

by cultured human endothelial cells. Science 1985; 227(4693): 1477-1479.

Galbraith CG, Skalak R and Chien S. Shear stress induces spatial reorganization of the

endothelial cell cytoskeleton. Cell Motil Cytoskeleton 1998; 40(4): 317-330.

Kadohama T, Akasaka N, Nishimura K, Hoshino Y, Sasajima T and Sumpio BE. p38

Mitogen-activated protein kinase activation in endothelial cell is implicated in cell

alignment and elongation induced by fluid shear stress. Endothelium 2006; 13(1):

43-50.

Kuchan MJ and Frangos JA. Role of calcium and calmodulin in flow-induced nitric oxide

production in endothelial cells. Am J Physiol 1994; 266(3 Pt 1): C628-C636.

Levesque MJ and Nerem RM. The elongation and orientation of cultured endothelial cells

in response to shear stress. J Biomech Eng 1985; 107(4): 341-347.

McCann JA, Peterson SD, Plesniak MW, Wenstar TJ and Haberstroh KM. Non-uniform

flow behavior in a parallel plate flow chamber : alters endothelial cell responses.

Ann Biomed Eng 2005; 33(3): 328-336.

Smart EJ, Ying Y, Donzell WC and Anderson RG. A role for caveolin in transport of

cholesterol from endoplasmic reticulum to plasma membrane. J Biol Chem 1996;

271(46): 29427-29435.

Sun RJ, Muller S, Stoltz JF and Wang X. Shear stress induces caveolin-1 translocation in

cultured endothelial cells. Eur Biophys J 2002; 30(8): 605-611.

68

Traub O and Berk BC. Laminar shear stress: mechanisms by which endothelial cells

transduce an atheroprotective force. Arterioscler Thromb Vasc Biol 1998; 18(5):

677-685.

Tricot O, Mallat Z, Heymes C, Belmin J, Leseche G and Tedgui A. Relation between

endothelial cell apoptosis and blood flow direction in human atherosclerotic

plaques. Circulation 2000; 101(21): 2450-2453.

Topper JN and Gimbrone MA Jr. Blood flow and vascular gene expression: fluid shear

stress as a modulator of endothelial phenotype. Mol Med Today 1999; 5(1): 40-

46.

Wang JH, Goldschmidt-Clermont P, Wille J and Tin FC. Specificity of endothelial cell

reorientation in response to cyclic mechanical stretching. J Biomech 2001; 34(12):

1563-1572.

Wojciak-Stothard B and Ridley AJ. Shear stress-induced endothelial cell polarization is

mediated by Rho and Rac but not Cdc42 or PI 3-kinases. J Cell Biol 2003; 161(2):

429-439.

Yang Z, Tao J, Wang JM, Tu C, Xu MG, Wang Y, Chen L, Luo CF, Tang AL and Ma H.

Fluid shear stress upregulated endothelial nitric oxide synthase gene expression

and nitric oxide formation in human endothelial progenitor cells. Zhonghua Xin

Xue Guan Bing Za Zhi 2007. 35(4): 359-362.

Young EW, Wheeler ER, and Simmons CA. Matrix-dependent adhesion of vascular and

valvular endothelial cells in microfluidic channels. Lab Chip 2007; 7(12): 1759-

1766.

69

CHAPTER 3

Real-time kinetic analysis of platelet function in a flow

chamber with matrix proteins covalently attached onto a

polydimethylsiloxane surface.

70

Introduction

Platelet adhesion and aggregation is the first step in the physiological defence mechanism

of the body in blood vessel injury. Exposure of extracellular matrix at the sites of sub

endothelial disruption leads to the capture of platelets to the matrix through various

receptors including GPIb-IX-V and GPVI, resulting in platelet activation, followed by

thrombi formation. Shear forces generated by blood flow play an important role in

platelet adhesion and hence vascular hemostasis.

Several investigations on platelet function have focused on the platelet adhesion to

various matrices (fibrinogen, collagen) under flow conditions. In all of these studies, glass

slides are coated with fibrinogen, collagen or vWF etc (Turner et al 2001; Remijn et al

2002)

and then used to study platelet aggregation mechanisms. Numerous devices

including parallel plate flow chambers, which mimic the in vivo conditions (Goncalves et

al 2003), are available for platelet aggregation studies under various physiological

conditions. Some of these flow devices and microfluidic devices have been custom

laboratory prototypes, while others are commercially available. There is currently little to

no availability of simple, reliable, robust, and inexpensive flow chambers for use in

kinetic aggregation studies.

Table 1 – Devices available to study platelet aggregation in vitro.

Device Advantages Disadvantages Kinetic/Static

Cone and plate

viscometer

High shear rates can

be attained

Real time analysis

not possible

Static

Parallel plate flow

chamber

Easy to use and

manufacture,

Cannot be easily

modified, requires

sizable volumes of

reagents

Kinetic

Microfluidic devices Real time imaging,

less sample volumes

and cells required

Costly Kinetic

71

In the present study, we describe the construction and testing of a parallel plate flow

chamber, which fits onto an inverted microscope and permits the optical monitoring of

the binding of fluorescently-labelled platelets, in whole blood, onto platelet-binding

proteins covalently attached to a small area (~0.8 mm2) on the flow-device’s surface

under in vivo-like conditions. The flow device was validated by comparing the kinetics

of adhesion of resting as well as Ca2+

- and ADP-activated platelets from normal and type

2 diabetic subjects onto fibrinogen and collagen covalently attached to the flow devices.

72

Experimental methods

Subject selection

Healthy human subjects (n=5), ages 25-40 years were chosen to participate in the study

only if they showed no overt symptoms of disease and were taking no medication.

Diabetic human subjects, ages 25-40 (n=5) on diet therapy alone and achieving stable and

satisfactory glycemic control (fasting glycemia and glycosuria variation <15%; post-

prandial glycemia variation<25% and HbA1c<7.5%) were chosen for inclusion in the

study. None of the patients smoked, had history of alcohol abuse or were taking insulin or

any drugs known to lower lipids or interfere with the coagulation and antioxidant

systems. The experimental protocols were approved by the University of Windsor

Research Ethics Board.

Construction of flow chamber

The flow chamber consisted of a Teflon boundary sealed with 1.5cm microscope

coverslips (Figure 1 A). The walls of the flow cell were made of polydimethylsiloxane

(PDMS) (Sylgard 184, Dow Corning). A layer of PDMS, ~ 0.96m thick, was poured on

the bottom of the chamber.

Plasma Oxidation of PDMS

To generate silanol groups on inert PDMS (Miyaki et al 2007) (Figure 1 B), plasma

oxidation was carried out using a Plasma cleaner PDC-32G (Harrick Plasma, USA).

Protein immobilization chemistry

After plasma oxidation, 0.2µl of 2% aminopropyltrimethoxysilane (APTMES) (Sigma,

Canada) was added on ~1 mm2

surface of PDMS, in the centre of the flow chamber. After

10 minutes, a 0.5 µl portion of 0.5mM disuccinimidyl suberate (DSS) (Pierce, USA)

solution and 5M of Type I fibrinogen from bovine plasma (Sigma) solution was added

to the APTMES dot in the geometrical centre of the flow chamber. For the experiments

with collagen immobilization, 8M of collagen type I from rat tail (BD Biosciences) was

used. The reaction was stopped after 15 minutes by adding Tris buffer pH 8. The

immobilization chemistry is shown in Figure 1 B.

73

Aminolysis of succinimidyl group

DSS has N-hydroxysulfosuccinimide (NHS) esters at each end and during the

crosslinking reaction of DSS with primary amines, succinimidyl group is aminolyzed and

released. The kinetics of the crosslinking reaction was determined by changes in

absorption by succinimidyl group at 260 nm. The reaction was performed in Tris buffer

(pH 8) with aminosilane alone, aminosilane and DSS, fibrinogen only, fibrinogen and

DSS, collage only or collagen and DSS. All measurements were taken using Agilent 8453

UV-Visible spectrophotometer.

FITC-Fibrinogen labelling and standardization of cross linking time

Fibrinogen was incubated with fluorescein-isothiocyanate (FITC) at room temperature for

2 h in the presence of 1 M bicarbonate buffer pH 9.0. The labelled fibrinogen was run

over G25 column in the dark to remove unbound FITC. Protein concentration was

determined by bicinchoninic acid (BCA) assay. Aminosilane was added to plasma

oxidized PDMS surface as described earlier. FITC-fibrinogen was added onto

aminosilane either with DSS or after 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 sec of DSS

addition. After 15 min incubation in dark, excess fibrinogen was washed off with Tris

buffer and the bound FITC-fibrinogen was detected by Zeiss Axiovert 200 fluorescence

microscope.

SDS washing of immobililized fibrinogen

Fibrinogen was fluorescently labelled with FITC and immobilized on PDMS by the

method described above. Fibrinogen-FITC adsorbed on PDMS was used as a control.

This was followed by addition of 2% SDS and the samples were incubated in dark for 3

hours. After incubation, the PDMS surface was washed with PBS and images were taken

using Zeiss Axiovert 200 fluorescence microscope.

74

Blood collection and washing of platelets

Platelets were isolated as described previously (Miersch et al 2007) and were labelled

with 60 μM BODIPY® FL N-(2-aminoethyl) maleimide (Molecular Probes, Canada).

After 30 min incubation, platelets were washed twice with HEPES-ACD buffer to remove

the excess dye and were put back into whole blood.

Light transmission platelet aggregometry

HEPES ACD was added to BODIPY® FL N-(2-aminoethyl) maleimide labelled and

unlabelled platelets to get a final concentration of 1 X 107 platelets/mL. 5µM ADP was

added as an agonist after ~ 40 sec. The platelet samples without ADP were used as

controls. The kinetics of the aggregation reaction were determined by measuring

absorbance at 600 nm for 5 min.

Perfusion studies in flow chambers

The flow cell circuit used for this investigation consists of a custom designed flow

chamber, PTFE Teflon sterile tubing (0.031” X 0.062”), a medium reservoir, and a

peristaltic pump (Reglo Digital MS 4/6, model ISM 833, Ismatec).

Statistical analysis

Data is expressed as an average of all trials and statistical analysis was done by student’s t

test.

Chemical structures in Figure 1 were drawn with ChemDraw 11.0 (Cambridge Software).

75

RESULTS

Flow chamber geometry and immobilization chemistry

The conventional flow chambers test platelet adhesion and aggregation by coating

fibrinogen/collagen on glass slides. In the present study, we plasma-activated an

otherwise inert matrix PDMS and used a bifunctional, primary amine-directed crosslinker

DSS, to immobilize platelet-binding proteins on the PDMS surface (Figure 1B).

76

Figure 1 – (A) Geometry of the flow chamber used for perfusion studies. (B) Chemistry

of immobilizing fibrinogen and collagen on PDMS. Hydroxyl groups were formed on

inert and hydrophobic PDMS by plasma oxidation. Coating of APTMES on polymer

surface after plasma oxidation resulted in an amine terminated surface layer. DSS was

used as a bi-functional crosslinker which reacts both with the amine terminated surface

layer and with the amine groups of the proteins to be immobilized.

77

Velocity profile

Figure 2 – Velocity profile. The graph shows the velocity profile from the inlet to the

two different points in the centre of the flow device. The velocity of flow is zero in the

boundary layer and rises monotonically with distance z from the wall, reaching maximum

at the centre. Z/Zr is the ratio of distance at a given point to the distance to the centre.

Vy/Vym is the ratio of the velocity at a given point to the velocity at the centre.

78

Crosslinking reaction between fibrinogen/collagen/aminosilane and DSS

DSS is a homobifunctional crosslinker which has amine reactive NHS esters at both ends.

It reacts with primary amines in slightly alkaline conditions to yield stable amide bonds.

This aminolysis reaction releases N-hydroxysuccinimide, which has an absorption

maximum at 260 nm. The aminolysis reaction between collagen and DSS and fibrinogen

and DSS is shown in Figure 3 A and B respectively.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 100 200 300 400 500 600

Ab

so

rba

nc

e a

t 2

60

nm

Time (in sec)

Aminosilane Aminosilane + DSS Collagen Collagen + DSS

A

79

Figure 3 – Kinetic data for aminolysis of DSS. Aminosilane, fibrinogen and collagen

were diluted in Tris buffer (pH 8) in different cuvettes. DSS was added after ~ 150 sec

and kinetics was measured at 260 nm using UV-Vis spectrophotometer (n=3). Samples

without DSS addition were used as controls.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 100 200 300 400 500 600

Ab

so

rba

nc

e a

t 2

60

nm

Time (in sec)

Aminosilane Amiosilane + DSS Fibrinogen Fibrinogen + DSS

B

80

Variations in time point at which fibrinogen/collagen is added to DSS affects

immobilization.

Since DSS is a homobifunctional crosslinker, if we add fibrinogen at a later point, all of

DSS might react with aminosilnae leading to less protein immobilization. To ensure

complete immobilization of fibrinogen onto PDMS surface, the time point at which

fibrinogen should be added to DSS has to be determined. FITC labelled fibrinogen was

added onto the DSS either with DSS or at different time points. The fluorescence was

maximum when DSS and fibrinogen were added together to aminosilane. There was no

fluorescence detected when only fibrinogen was added on PDMS surface without any

immobilization chemistry.

A

81

Figure 4 – Variations in time point at which fibrinogen/collagen is added to DSS

affects immobilization. (A) The images show FITC-fibrinogen immobilized on PDMS

surface where it was added onto aminosilane and DSS at varying time intervals. (B) The

bar graph shows average fluorescence intensity for FITC-fibrinogen fluorescence, where

each bar represents the mean ± SD from at least three independent experiments.

0

20

40

60

80

100

120

%

flu

ore

sc

en

ce

B

82

SDS washing of FITC-fibrinogen

Due to the hydrophobic nature of PDMS, it shows high affinity for proteins. In order to

show that the proteins (fibrinogen/collagen) are covalently linked and not adsorbed, the

PDMS surface was treated with SDS. Figure 5 illustrates that SDS washes off the protein

while about 90% of the protein remains on the PDMS-AS-DSS-Fib surface.

Figure 5 – SDS washing of FITC-fibrinogen. Fibrinogen was labelled with FITC and

immobilized on PDMS surface. The surface was incubated with 2% SDS for 3 hours and

was washed with Tris buffer. After 3 hours, the covalently linked protein was still there

whereas the control showed almost no protein left (n=3).

83

Effect of bodipy labelling on platelet aggregation

The platelets were fluorescently labelleled with BODIPY FL N-(2-Aminoethyl)

maleimide for the further experiments. To analyze the effects of the fluorescent label on

platelet aggregation, light transmission aggregometry was done using UV-Vis

spectrophotometer. Absorbance at 600 nm was monitored for labelled and unlabelled

platelets in the presence and absence of ADP. As can be seen from the optical assessment

of aggregation shown in Fig. 6, bodipy-modification at the levels we employ does not

interfere with ADP-induced aggregation.

Figure 6 – Effect of bodipy labelling on platelet aggregation. The platelets were

diluted in HEPES ACD and 5 µM ADP was added as an agonist after ~ 40 sec. The

decrease in absorbance indicates platelet aggregation. The platelets without ADP addition

were used as control.

1.48

1.49

1.5

1.51

1.52

1.53

1.54

1.55

1.56

0 50 100 150 200 250 300 350

Ab

so

rban

ce

a

t 6

00

nm

Time (in sec)

ControlNormal

ADPNormal

ControlBodipy

ADPBodipy

84

Validation of the flow chambers

In order to assess the suitability of the flow chamber and the immobilization chemistry for

platelet adhesion studies, adhesion to collagen and fibrinogen was tested. Flow chambers

were placed onto an inverted fluorescence microscope (Zeiss Axiovert 200M. Whole

blood containing fluorescently labelled platelets was perfused over the flow chambers

containing either PDMS alone or PDMS-immobilized BSA or fibrinogen or collagen. The

perfusion rate was 2 ml/min, which generates shear equivalent to that of descending aorta

(5 dyne/cm2). The platelets were either activated by the injection of Ca

2+ (1 mM) or ADP

(20 µM) at t=0. The raw image data, at 40 s intervals, is presented in Figure 7. With the

controls, PDMS alone or PDMS-immobilized BSA, there were <50 platelets attached

after 200 s of perfusion (Figure 7).

85

86

Figure 7 – Comparison of platelet aggregation in normal and type 2 diabetic

subjects. Whole blood containing fluorescently labelled platelets was passed over PDMS

alone and PDMS with immobilized BSA, fibrinogen (Fib) or collagen (Coll) using either

1 mM calcium (Ca2+

) or 20 µM ADP as activators. The images show time dependent

increase in platelet adhesion to fibrinogen and collagen, with very little adhesion to the

controls (PDMS and BSA). The total number of adherent platelets after 200 sec is greater

in the case of type 2 diabetic (T2D) subjects as compared to normal (N) subjects,

irrespective of the protein/activator used. All experiments were done at 37oC. Images

were captured in 20 sec intervals by Zeiss Axiovert 200 microscope with achromat 5X

objective (Carl Zeiss), equipped with a Retiga EX cooled monochrome 12 bit camera (Q

imaging) and an Xcite series 120 (EXFO,Canada) mercury lamp. Image capture and the

quantification of the adhered platelets were facilitated by Northern Eclipse software

(Empix, Canada).

87

Kinetic plots of platelet aggregation

Images over the immobilized protein (fibrinogen or collagen) field were captured every

20 s. The platelets were counted in each frame using Northern Eclipse software and the

kinetic plots were constructed (Figure 8). There is an increased rate of platelet

aggregation in diabetic subjects as compared to the controls.

Figure 8 – Kinetic plots of platelets adhered to immobilized fibrinogen and collagen

over time. Blood reconstituted with fluorescently labelled platelets from normal and

diabetic subjects was perfused through the flow chambers containing immobilized

fibrinogen and collagen. Graphs (i) and (ii) show platelets adhered to collagen and

fibrinogen, respectively, over time, when calcium was used as an activator. Graphs (iii)

and (iv) show increasing number of platelets adhered to collagen and fibrinogen

respectively, when ADP was used as an activator. The number of platelets adhering over

time is greater in diabetic subjects (squares) as compared to normal subjects (triangles).

The platelet binding data is extracted from the entire image data set taken at 20 sec

intervals. Each point represents the mean ± SD from at least three independent

experiments. The solid lines represent the best fit line for the first order treatment of the

binding data: Y=A (1-e-kt

).

88

The total number of platelets bound to the protein surfaces are summarized in Table 1. In

general, the maximum number of platelets bound at saturation (~200 s) was larger for

platelets from T2D subjects in comparison to those from the normal subjects, under all

conditions examined. The largest ratio between T2D to normal of ~1.73 was obtained

with Ca2+

-activated platelets on a fibrinogen surface. The smallest ratio of ~1.31 was

obtained with ADP-activated platelets on a collagen surface.

Table 1 – The table shows the total number of normal and type 2 diabetic (T2D) platelets

± SD attached to fibrinogen (Fib) and collagen (Coll) after 200 sec, with calcium (Ca2+

)

and ADP as activators.

89

Table 2 – The first order rate constants were calculated from the kinetic plots of the

platelet adhesion data. The T2D platelets have higher rate constants as compared to the

normal (*P<0.05).

90

Discussion

The novel aspect of this study is the surface chemistry, which can be utilized for facile

patterning of immobilized proteins on PDMS surface. The design of the flow device and

immobilization chemistry introduced here can easily be adapted to microfluidic/lab on

chip applications for the analysis of hemostasis. The glass coverslip on the bottom of the

flow chamber can be removed, which allows for cheap and reproducible flow chambers

for further experiments.

The washing of PDMS with SDS could not remove the immobilized fibrinogen.

This indicates that AS-DSS can bind protein more tightly than physiosorption. Light

transmission aggregometry illustrated that platelet labeling with Bodipy does not affect

the aggregation of platelets in response to agonists’.

When the whole blood was passed over fibrinogen or collagen coated flow

devices there was a continuous build up in the adhered platelets over the initial density

pattern established in the previous time frames indicating that the interaction was

irreversible. At the end of the binding experiments ~10 mL of buffer was pumped through

the flow chambers. The number of adhered platelets did not change, which was a further

indication of the irreversibility of the interaction with protein matrices covalently attached

to the PDMS surface. One observation from the kinetic plots of the data was that when

platelets were activated with Ca2+

, with either on the collagen or the fibrinogen surface,

there was a 20 s to 40 s lag prior to the initiation of platelet binding irrespective of the

platelet source (T2D or normal) (Figure 8 i, ii). In contrast, this lag was absent in the

ADP-activated platelets (Figure 8 iii, iv).

It is well established that in T2D, the platelets have altered in vitro adhesion and

aggregation patterns and are hypersensitive to agonists as compared to the normal

platelets (Glassman et al 1993; Dittmar et al 1994; Haouari et al 2008; Vinik et al 2001).

Therefore, an important step in the validation process was to determine whether our flow

devices were able to discern functional differences between platelets from normal and

T2D subjects. To this end, we compared the adhesion kinetics of Ca2+

or ADP activated

platelets from control and T2D subjects onto immobilized collagen and fibrinogen in the

flow chambers. A qualitative examination of the raw data (Figure 7) revealed that

91

platelets from T2D subjects gave rise to more platelet adhesion irrespective of the

activator used or the immobilized surface. These observations were elegantly

substantiated upon kinetic treatment of the data (Fig 8 i to iv and Tables 1 and 2).

The rate constants extracted from first order kinetic treatment of the data (Table 2)

revealed that the binding rate constants for platelets from normal subjects were in general

independent of the activator used or the protein immobilized since the differences

between the rates (0.014 0.002 sec-1

) were not statistically significant. However, when

the rate constants of the various activator/protein surface combinations from T2D were

compared to normal, in all cases the T2D gave rise to 1.12-fold to 1.33-fold larger and

statistically significant, platelet-binding rate constants (0.018 0.002 sec-1

).

Conclusions

In conclusion, here we have introduced a simple and robust method for the construction

of flow cells made out of PDMS as well as the chemistry for the covalent attachment of

any platelet-reactive peptides or proteins onto the PDMS surface. We have also

demonstrated that the flow cells can readily provide real time data on the kinetics of

platelet binding and can detect subtle pathological changes in platelet function introduced

as a result of pathologies such as type 2 diabetes.

92

References

Dittmar S, Polanowska-Grabowska R and Gear AR. Platelet adhesion to collagen under

flow conditions in diabetes mellitus. Thrombosis Research 1994; 74: 273-83.

Glassman AB. Platelet abnormalities in diabetes mellitus. Ann Clin Lab Sci. 1993; 23:

47-50.

Goncalves I, Hughan SC, Schoenwaelder SM, Yap CL, Yuan Y and Jackson SP, Integrin

alpha IIb beta 3-dependent calcium signals regulate platelet-fibrinogen

interactions under flow. Involvement of phospholipase C gamma 2. J Biol Chem

2003; 278: 34812-34822.

Haouari M El and Rosado JA. Platelet signaling abnormalities in patients with type 2

diabetes mellitus: a review. Blood Cells Mol Dis. 2008; 41: 119-123.

Miersch S, Sliscovic I, Raturi A and Mutus B. Antioxidant and antiplatelet effects of

rosuvastatin in a hamster model of prediabetes. Free Radical Biology & Medicine

2007; 42: 270–279.

Miyaki K, Zeng HL, Nakagama T and Uchiyama K. Steady surface modification of

polydimethylsiloxane microchannel and its application in simultaneous analysis of

homocysteine and glutathione in human serum. Journal of Chromatography.A.

2007; 1166: 201-206.

Remijn JA, Wu YP, Jeninga EH, Jsseldijk MJW, Willigen GV, Groot PG, Sixma JJ,

Nurden AT and Nurden P. Role of ADP Receptor P2Y12 in Platelet Adhesion and

Thrombus Formation in Flowing Blood. Arteriosclerosis,Thrombosis, and

Vascular Biology 2002; 22: 686-691. Turner NA, Moake JL and McIntire LV Blockade of adenosine diphosphate receptors

P2Y12 and P2Y1 is required to inhibit platelet aggregation in whole blood under

flow. Blood 2001; 98: 3340-3345.

Vinik AI, Erbas T, Park TS, Nolan R, Pittenger GL. Platelet dysfunction in type 2

diabetes. Diabetes Care 2001; 24: 1476-1485.

93

CHAPTER 4

Whole blood, flow-chamber studies in real-time indicate a

biphasic role for thymosin β-4 in platelet adhesion

Published as original research as Kaur et al. 2010

94

Introduction

Thymosin β4 (Tβ4) is a water-soluble, ubiquitous, highly conserved 43-amino acid acidic

polypeptide (pI 5.1) with a molecular weight of 4.9 kDa (Low et al 1981, 1982; Yu et al

1994; Goodall et al 1983, 1983). It was originally isolated from the thymus but is present

in different concentrations in different tissues, cell types and extracellular fluids (blood,

plasma, saliva, tears). It is found at concentrations of ~5 nM to ~80 nM in plasma to ~500

μM in platelets (Hannappel et al 1982, 1985, 1987; Huff et al 2001). Tβ4 lacks a

secretion signal and hence its presence in extracellular fluids could be due to cellular

damage.

The major intracellular function of Tβ4 in mammalian cells is to form a 1:1

complex with G-actin, preventing its polymerization (Low et al 1981; Hannappel et al

1985; Safer et al 1991). Apart from its interactions with actin, Tβ4 is capable of

modulating a wide array of biological functions including tissue (Goldstein et al 2005)

and wound repair (Sosne et al 2002) as well as anti-inflammatory (Young et al 1999)

antiapoptotic and neurotrophic (Sun et al 2007) properties. Tβ4 has been also shown to

play a relevant role in angiogenesis (Smart et al 2009) and neural development (Lin et al

1990). It also protects the cells against oxidative damage and upregulates anti-oxidative

enzymes catalase and SOD (Ho et al 2008). The role of Tβ4 in increasing hair growth and

dermal repair has also been studied, where it promotes stem cell migration and their

subsequent differentiation into keratinocytes and hair follicles (Philip et al 2004).

The ability of Tβ4 to affect these diverse biological functions has been ascribed to

an array of short, bioactive, peptide sequences within the Tβ4-primary sequence (Sosne et

al 2010) (Fig. 1).

95

Figure 1 – Location and activity of different peptides derived from Tβ4 (Image

taken from Sosne et al 2010).

96

The largest concentrations of Tβ4 are found in leukocytes and platelets (Low et al 1981;

Yu et al 1994; Goodall et al 1983; Hannappel et al 1985, 1987). In resting platelets, it has

been estimated that ~60% of the actin is prevented from polymerizing by complexing

with Tβ4 (Fox 1993). Upon platelet activation, Tβ4 is secreted and cross-linked

enzymatically by factor XIIIa (a transglutaminase) to fibrin in a time- and Ca2+-

dependent

manner (Huff et al 2002; Makogoneko et al 2004) thus increasing the local concentration

of Tβ4 near sites of clots and tissue damage, where it is postulated to contribute to wound

healing, angiogenesis and inflammatory responses. Platelets carry and release large

amounts of Tβ4. Yet the role of Tβ4 on platelet thrombus formation has yet to be fully

investigated. The current study represents a small step towards this goal. Here, we have

tested the effects of Tβ4 extraneously added to whole blood, on deposition of ADP

activated platelets onto fibrinogen under conditions of continuous flow and shear.

97

Experimental methods

Subject selection

Healthy human subjects (n=5), ages 25–40 years were chosen to participate in the study

only if they showed no overt symptoms of disease and were taking no medication. The

experimental protocols were approved by the University of Windsor Research Ethics

Board.

Construction of the flow chamber

The flow chamber consisted of a Teflon boundary with matching holes (1.75mm) drilled

through sides a and b (Fig. 2A). The Teflon boundary was first attached onto 1.5 cm

microscope coverslips by painting a small amount of Sylgard 184 (trade name of

polydimethylsiloxane- PDMS), (Dow Corning) base plus curing agent (mixed in a 10:1

ratio). Teflon® tubing (2 cm) was then inserted through the holes and enough Sylgard

184, (Dow Corning) base plus curing agent was poured to fill the mold. After curing for 6

h at 60 °C the Teflon tubing was removed by pulling through the holes (sides a or b, Fig.

2A). The flow chamber was then placed into a plasma cleaner PDC-32G (Harrick Plasma,

USA) in order to generate silanol groups (Miyaki et al 2007) on the otherwise inert

PDMS-walls of the flow chamber (Fig. 2B).

Protein immobilization onto plasma activated PDMS surfaces in the flow chambers

After plasma oxidation, 5 μl of 2% aminopropyltrimethoxysilane (APTMES) (Sigma,

Canada) was added to the geometric centre of each of the flow channel with the aid of a

Hamilton syringe. After 10 min, a 0.5-μl portion of 0.5 mM disuccinimidyl suberate

(DSS) (Pierce, USA) solution was added to the APTMES ring at the centre of the flow

channels. Within 30 s of DSS addition, either 1 μl of either Type I fibrinogen (5 μM)

from bovine plasma (Sigma-Aldrich, Canada) or of collagen type I (8 μM) from rat tail

(BD Biosciences) was introduced into the reaction mixture. The reaction was stopped

after 15 min by pumping 10 mL of Tris–HCl buffer (0.15 M, pH 8.0) at a rate of 2 ml/min

through the flow chamber.

98

Blood collection and washing of platelets

Platelets were isolated as described previously (Miersch et al 2007) and were

fluorescently labelled by reacting for 30 min, with 60 μM BODIPY® FL N-(2-

aminoethyl) maleimide (Molecular Probes, Canada). The labelled platelets were

harvested by 2 centrifugation-wash (HEPES-ACD buffer) steps to remove the excess dye

and were reintroduced into the whole blood sample that they were originally isolated

from.

Monitoring platelet deposition via flow cells

A flow cell with the coverslip face-down was placed in the turret of a Zeiss Axiovert 200

M inverted fluorescence microscope equipped FITC filter cube, and 5×objective.

Fluorescence images were captured at 20-s intervals with the aid of a CCD camera

(Hitachi KP-F140F). Two syringe pumps (New Era Pumps, NE-300) were utilized to mix

the activating agent (ADP, 400 μM/ Ca2+

, 100mM; flow rate 0.05 mL/min) with the

whole blood containing the fluorescently-labelled platelets (flow rate 0.95 mL/min) at a t-

junction. This yields final Ca2+

and ADP concentrations in the flow cell of 5 mM and 20

μM, respectively. At the start of the experiment only the blood was pumped through the

channel to adjust the focus and to optimize image capture parameters. At t=0 the

activating agent pump was started and image capture initiated. A given blood sample was

pushed separately though each flow channel thus generating 4 sets of binding data per

sample. The estimated shear on the wall of the flow cells was 150 s−1

(5 dyne/cm2), which

represents shear rate in the descending aorta or veins. Image analysis was performed with

the aid of Northern Exposure 6.0 (Empix, Mississauga, ON) and ImageJ

(NIH) imaging software packages.

Tβ4 binding assay

A 96-well plate was filled with 50 μL of PDMS at a ratio of 10:1, base: curing agent.

Fibrinogen was immobilized on PDMS as described before. Tβ4 was labelled with eosin-

isothiocyanate (EITC) at room temperature for 2 h in the presence of 1 M sodium

bicarbonate buffer pH 9.0. The labelled Tβ4 was run over a G25 column in the dark to

remove unbound EITC. Protein concentration was determined by bicinchoninic acid

99

(BCA) assay. Tβ4 was added to the wells at concentrations of 0.1 μM, 0.2 μM, 0.5 μM, 1

μM, 2 μM and 4 μM. In some wells, unlabelled 4 μM Tβ4 was also added. For control,

labelled Tβ4 was added to the activated PDMS alone. The plate was incubated under

gentle agitation for 2 h in the dark. Fluorescence was monitored in the solution before and

after incubation by Cary Eclipse Fluorescence Spectrophotometer with excitation at 520

nm and emission at 540 nm. To study the thymosin beta 4 binding to fibrinogen under

flow, fibrinogen was immobilized in the flow chamber (i.e. the yellow region Fig. 1A)

and excess eosin-Tβ4 was introduced onto the fibrinogen. The flow chamber was then

washed with PBS to remove excess eosin-Tβ4. Increasing concentrations of unlabelled

Tβ4 (10 nM–1 μM) were pumped through the flow cells with a syringe pump (New Era

Pumps, NE-300) at a rate of 1.0 mL/min and 0.2 mL fractions were collected using a

fraction collector (Bio-Rad, Model 2110). Fluorescence was monitored in the fractions by

Cary Eclipse Fluorescence Spectrophotometer with excitation at 520 nm and emission at

540 nm.

Statistical analysis

Data is expressed as an average of all trials and statistical analysis was performed by

Student's t-test the error bars represent s.d. Chemical structures in Fig. 1 were drawn with

ChemDraw 11.0.

100

Results

Flow chamber geometry and immobilization chemistry

Figure 2 – Geometry of the flow chamber and chemistry used for immobilizing

fibrinogen/collagen in the flow chamber.

101

Validation of the flow chambers

In these studies, the raw image files collected at 20 s intervals were converted to 8-bit

gray scale images. We then used the ImageJ plugin, Multi Otsu Threshold introduced by

Dos Santos et al. (Santos et al 2009) to ensure that we did not count the platelets that

were transiently associated with the immobilized proteins (appear as streaks in the raw

images: indicated with arrows in Fig. 3). Otsu multi-thresholding eliminated the streaks as

well as the out of focus platelets deposited on the edges (see Fig. 2 a, b, c, d, e vs. a', b', c',

d', e'). The processed-images were subsequently counted by using ImageJ: Step 1

menu>image>adjust>threshold; and Step 2—Menu>analyze>analyze particles.

102

Figure 3 – Demonstration of the Otsu Multi-Thresholding ImageJ plugin to exclude non-

deposited, transiently associated platelets from being counted in the deposition data.

Representative raw images obtained at 120 s in flow cells with immobilized BSA, a;

immobilized collagen with Ca2+

as activator, b; immobilized collagen with ADP as

activator, c; immobilized fibrinogen with Ca2+

as activator, d; immobilized fibrinogen

with ADP as activator, e. The same Images after Otsu Multi Thresholding with

immobilized BSA, a'; immobilized collagen with Ca2+

as activator, b'; immobilized

collagen with ADP as activator, c'; immobilized fibrinogen with Ca2+

as activator, d';

immobilized fibrinogen with ADP as activator, e'. Arrows point to non deposited platelets

appearing as streaks.

103

Platelet deposition on collagen/fibrinogen in the presence of Ca2+/

ADP

A representative set of platelet deposition data from blood samples isolated from the same

subject, exposed to the either Ca2+

or ADP as activator with either BSA or fibrinogen or

collagen immobilized into the flow cell surfaces is presented in Fig. 4 A – F. The platelet

deposition data for collagen and fibrinogen were well accommodated by a first order

kinetic process [# of platelets deposited at a given time (t) =Maximum # of platelets

deposited*(1- e−k

obs*t

)]. The rate constants estimated from first order kinetic treatment of

the data revealed that the rate of platelet deposition was independent of both the activators

used (Ca2+

or ADP) and the protein immobilized (collagen or fibrinogen) since the

differences in the rates obtained under the various conditions were not statistically

significant (Fig. 4G). This same trend was observed with all subjects tested (n=5).

104

A

B

C

D

E

F

G

H

105

Figure 4 – Kinetic plots obtained from particle counts of Otsu Multi Threshold images

for platelets deposited onto immobilized BSA, A; immobilized collagen with Ca2+

as

activator, B; immobilized fibrinogen with Ca2+

as activator, C; immobilized

fibrinogen/collagen without any activator, D; immobilized collagen with ADP as

activator, E; immobilized fibrinogen with ADP as activator, F; (n=4 from a single

subject). Composite rate constants: an average rate constant from each subject was

obtained by performing the deposition experiment under each condition (A to F) in

quadruplicate. The average value for each condition was then averaged for all the subjects

(n=4), G. Composite # of deposited platelets: an average # of deposited platelets from

each subject were determined from the 120 s images by performing the deposition

experiment under each condition (A to F) in quadruplicate. The average value for each

condition was then averaged for all the subjects (n=4), H. The solid lines represent the

best fit line for the first order treatment of the binding data: Y = A (1- e−kt

). Error bars

represent s.d.

106

The effect of Tβ4 on platelet deposition under conditions of flow.

The flow system was then employed to test the effect of Tβ4-dose on the deposition of

ADP-activated platelets to fibrinogen immobilized flow cells. In these studies, Tβ4, at the

concentrations indicated (Fig. 5) was incubated for 15 min with the blood/fluorescently

labelled platelet mixtures before the deposition experiments were carried out. As can be

seen from the kinetic binding data (Fig. 5A) and a representative data set of the images

captured at 120 s from the blood samples obtained from the different subjects (S1 to S4,

Fig. 5B), Tβ4 had a biphasic effect on both the rates of deposition as well as the number

of platelets deposited. Above a critical concentration of ~0.2 μM Tβ4 the platelet

deposition is not uniform over the flow cell surface as observed in the control or the

validation experiments (Fig. 3 and 4). Instead, large thrombi are evident indicative of

platelet–platelet interactions. These are reflected in the ~4-fold increase in the estimated

number of platelets deposited (Fig. 5D). The estimated rate constants of platelet

deposition also increased ~1.5-fold during this process.

107

A

B

108

Figure 5 – The effect of Tβ4-dose on the rate and density of platelet deposition.

(A) Kinetic plots obtained from particle counts of Otsu Multi Thresholded images for

platelets deposited on immobilized fibrinogen with ADP as activator, as a function of

Tβ4-dose. The solid lines represent the best fit line for the first order treatment of the

binding data: Y = A (1- e−kt

). Error bars represent s.d. (n=4). (B) Representative raw

images from each subject (S1 to S4) at 120 s as a function of Tβ4-dose. (C) Composite

rate constants: an average rate constant from each subject was obtained by performing the

platelet deposition as a function of Tβ4-dose, in quadruplicate. The average value for

each [Tβ4] was then averaged for all the subjects. Error bars represent s.d. (n=4). (D)

Composite # of deposited platelets: an average # of platelets deposited from each subject

was determined from the 120 s images as a function of Tβ4-dose, in quadruplicate. The

average value for each [Tβ4] was then averaged for all the subjects. Error bars represent

s.d. (n=4).

C

D

109

Tβ4 binding to immobilized fibrinogen

The decrease in platelet aggregation in the presence of high concentrations of Tβ4

suggests that Tβ4 might be binding with fibrinogen and hence inhibiting fibrinogen

mediated platelet aggregation. This led us to propose this hypothesis that Tβ4 might have

a binding site on fibrinogen. In order to test our fibrinogen binding hypothesis, we

immobilized fibrinogen onto PDMS on the bottom of 96-well plates using the same

chemistry as in the flow cells (Fig. 1). We then used a fluorescent Tβ4 derivative, eosin-

Tβ4, to perform equilibrium binding studies. In these experiments increasing amounts of

eosin-Tβ4 were added to either activated PDMS (Fig. 6A, diamonds) or PDMS-

fibrinogen (Fig. 6A, triangles) or PDMS-fibrinogen containing a constant amount of

unlabelled Tβ4 (Fig. 6A, circles). There was only significant Tβ4-binding to fibrinogen

immobilized PDMS and not to PDMS alone or when labelled Tβ4 was added to PDMS-

fibrinogen in the presence of a constant amount of unlabelled Tβ4. These experiments

indicate that we are observing a specific interaction between fibrinogen and eosin-Tβ4.

The KD estimated for this interaction was ~126±18 nM (i.e. negative reciprocal of the

slope of the Scatchard plot of the binding data, Fig. 6B).We also estimated the Tβ4-

fibrinogen interaction under flow. To do this, excess eosin-Tβ4 was introduced onto the

fibrinogen immobilized in the flow chambers (i.e. the yellow region Fig. 2). The excess

eosin-Tβ4 was removed by pumping 2 mL of PBS over the flow cells. Next increasing

amounts of unlabelled Tβ4 was pumped through the flow cells with a syringe pump at a

rate of 1.0mL/min and 0.2 mL fractions were collected. The total fluorescence (eosin-

Tβ4) summed over fractions 1 and 2 was plotted against unlabelled [Tβ4] (Fig. 6C). The

data were fitted to the competitive binding equation Eq. (1)

100% (total fluorescence) - % fluorescence eluted f ([½Tβ4])) =

[eosin−Tβ4]

KD-eosin−Tβ4 (1 + [Tβ4]/ KD-Tβ4 ) + (eosin−Tβ4)

where KD− eosin-Tβ4 = the dissociation constant between fibrinogen and eosin-Tβ4; and KD−

Tβ4 = the dissociation constant between fibrinogen and unlabelled-Tβ4. The KD− Tβ4

estimated under flow conditions was 66±20 nM.

110

Figure 6 – Binding of eosin-Tβ4 to fibrinogen immobilized on PDMS.

(A) Equilibrium binding of eosin-Tβ4 to fibrinogen. Increasing amounts of eosin-Tβ4

were added to activated PDMS (diamonds) or PDMS-fibrinogen (triangles) or PDMS-

fibrinogen containing a constant amount, 4 μM, of unlabelled Tβ4 (circles). Bound eosin-

Tβ4 fluorescence = total eosin-Tβ4 fluorescence−free eosin-Tβ4 fluorescence (i.e.

fluorescence of the supernatant after addition onto PDMS-wells) as a function of eosin-

Tβ4-dose. Error bars represent s.d. (n=5). (B) Scatchard plot of the binding data.

KD=−1/slope. Error bars represent s.d. (n=5). (C) Binding of eosin-Tβ4 to fibrinogen

under flow. Increasing concentrations of unlabelled Tβ4 were pumped over the flow cell

containing immobilized fibrinogen and eosin-Tβ4. The total fluorescence (eosin-Tβ4)

was plotted against unlabelled [Tβ4]. Error bars represent s.d. (n=3).

A

B

C

111

Discussion

In the present study, a new perfusion chamber to study platelet adhesion in vitro was

introduced. To validate the chamber, platelet deposition onto collagen and fibrinogen was

determined using Ca2+

and ADP as activating agents.

The average rate constant of platelet accumulation obtained under the various

conditions (immobilized collagen and fibrinogen with Ca2+

and ADP as agonists) was

0.026±0.0015 s−1

. This rate constant corresponds to a half-life of 27 s. This indicates that

the deposition process in the flow system is complete by ~130 s (i.e. 5× half-life). This

value corresponds well to 2 min to 6 min bleed times (i.e. the time it takes for a small skin

wound to stop bleeding) in normal humans (Schafer et al 2003). There were also no

statistically significant differences in the total number of platelets deposited to the flow

cell surfaces covered with fibrinogen or collagen which is indicative of the reproducibility

of the immobilization methodology employed here.

Platelets have high concentrations of Tβ4 and during blood clotting, Tβ4

concentration in the serum increases substantially. During blood coagulation, or by ADP

induced aggregation of platelets, Tβ4 is liberated from platelets and partially cross-linked

to fibrin by a transglutaminase, factor XIIIa. The covalent cross-linking of Tβ4 to the

fibrin clot might represent a mechanism to guarantee a high local concentration of the

peptide at the site of injury, probably supporting subsequent wound healing.

When Tβ4 was added to the blood, there was an increase in platelet deposition as

compared to the controls. However, at high concentrations of Tβ4, the platelet deposition

diminished. In terms of the half-life of deposition, this corresponds to a decrease from 27

s to 17 s (i.e. deposition is complete in ~85 s in the presence of Tβ4 as opposed to ~130 s

in its absence). Interestingly, as the concentration of Tβ4 reaches ~2 μM the deposition

rates and density return to near normal levels.

One possible explanation for Tβ4-mediated transient in platelet deposition could

be that Tβ4 has multiple effects on platelet thrombus formation. At low concentrations

Tβ4 could promote platelet activation by interacting with platelet receptors (ADP,

fibrinogen, etc.) converting them to high affinity states. Tβ4 could also bind to fibrinogen

112

in such a manner to prevent its interaction with platelet receptors, thus as Tβ4

concentrations rise, platelet deposition would be attenuated.

The binding of Tβ4 to fibrinogen and fibrin in the presence and absence of

transglutaminase was tested previously (Makogoneko et al 2004). In these studies,

fibrinogen was attached onto plastic, blocked and the amount of Tβ4-bound was

estimated by anti-Tβ4-antibodies conjugated to horse radish peroxidase. The

concentration range of Tβ4 employed was 5 μM to 30 μM. Although these authors did

observe the largest amounts of Tβ4 incorporation to fibrin in the presence of

transglutaminase, they did report a small amount of Tβ4-binding to fibrinogen

(Makogoneko et al 2004).

Our present result suggests that there is a high affinity Tβ4-binding domain on

fibrinogen that was previously undetected possibly owing to differences in the Tβ4-dose

or the binding assays employed. The fibrinogen binding ability of Tβ4 could explain the

biphasic response of Tβ4 on platelet deposition under conditions of flow as observed

here.

Furthermore, the results obtained in this study suggest a key modulatory role for

Tβ4 in thrombus formation. At low doses Tβ4 promotes platelet deposition and

aggregation by as yet unknown mechanism(s). But as platelets are activated and more

Tβ4 is released from platelets, its serum levels rise and prevent excessive thrombus

formation by binding to fibrinogen and preventing its interaction with platelets. It is also

very tantalizing to postulate that platelets hyperactivity in pathologies like type 2 diabetes

can result from deficiencies in intraplatelet Tβ4. The consequences of this would be that

more actin would be polymerized in the resting platelet and less Tβ4 would be released

into the blood during platelet activation to modulate the inhibitory phase by binding to

fibrinogen thus resulting in platelet hyperactivity.

113

Conclusions

These results suggest that Tβ4 could potentially increase the affinity of platelet receptors

for their ligands thus promoting platelet deposition. Tβ4 could also bind to fibrinogen and

as its concentration increased would prevent platelet–fibrinogen interactions resulting in

the attenuation of platelet deposition. This work suggests that Tβ4 might have a dual role

in platelet function.

114

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116

CHAPTER 5

Thymosin beta 4 alleviates endoplasmic reticulum stress in

retinal pigment epithelial cells

117

Introduction

Endoplasmic reticulum (ER) stress is caused by the accumulation of unfolded proteins in

the endoplasmic reticulum lumen. It has been implicated in the pathogenesis of many

neurodegenerative diseases including Alzheimer’s, Parkinson’s and Huntington’s disease

(Li et al 2008). All of these diseases are similar to age related macular degeneration

(AMD) in various pathological features. It has been recently proposed that ER stress

might have an important role in the pathogenesis of AMD (Salminen et al 2010). It has

also been shown to play a critical role in the early stages of diabetic retinopathy

progression (Li et al 2009). ER stress has been triggered by chronic ocular hypertension

in glaucoma mouse models, which leads to retinal ganglion cell death (Doh et al 2010).

The retinal pigment epithelium (RPE) is a monolayer of pigmented simple

cuboidal cells, which performs many functions essential for vision. The basolateral

membrane of the RPE faces the Bruch’s membrane and the apical membrane faces the

photoreceptor outer segments. RPE has many critical functions including transport of

nutrients (Vitamin A and C) from blood to retina, phagocytosis of the shedded

photoreceptor membranes, setting up the ion gradients within the interphotoreceptor

matrix and building up the blood-retina barrier. RPE dysfunction is central to the

development of AMD.

Age related macular degeneration (AMD) is the leading cause of loss of central

vision in elderly individuals. AMD is characterized by morphological and functional

abnormalities in RPE cells (Binder et al 2007). The pathogenesis of AMD involves the

buildup of drusen between Bruch’s membrane and RPE and accumulation of lipofuscin, a

pigmented aggregate of proteins and lipids, in RPE. Subretinal neovascularization, which

occurs due to VEGF overexpression in the RPE, is a major development of AMD.

Recently, it has been shown that ER stress causes increased expression of VEGF (Roybal

et al 2005).

Oxidative stress is a very important factor in the progression and onset of AMD

and can initiate ER stress in RPE (Beatty et al 2000, He et al 2008). It has been recently

proposed that ER stress might play an important role in the pathogenesis of AMD (Libby

et al 2010).

Thymosin 4 (Tβ4) is a water-soluble, 43 amino acid polypeptide with a

118

molecular weight of 4.9kDa (Low et al 1981; Yu et al 1994). It is a major G actin

sequestering protein in mammalian cells. Tβ4 has diverse biological roles including

angiogenesis, cell migration, tissue protection, and regeneration in skin, eyes and heart. It

has been previously reported that Tβ4 decreases inflammation and promotes wound

healing in corneal epithelial cells (Sosne et al 2002). Internalization of Tβ4 has been

reported in human corneal epithelial cells, HUVEC, bovine corneal endothelial cells and

human bone marrow-derived mesenchymal stem cells (Grant et al 1999; Ho et al 2007).

Herein we hypothesize that ER stress might play an important role in the RPE

damage during AMD and that Tβ4 can mitigate those effects. In the present study, we

report that Tβ4 can alleviate ER stress in retinal pigment epithelial cells.

119

Experimental methods

Cell Culture and treatment

Human retinal pigment epithelium cell line ARPE 19 was obtained from ATCC. Cells

were grown in 1:1 mixture of Dulbecco’s Modified Eagle’s Medium with Ham’s F-12

nutrient medium (DMEM F-12; ATCC, USA), 10% fetal bovine serum (FBS, Sigma,

Canada) and penicillin – streptomycin (Gibco, Canada). The cells were either plated on

coverslips in 35 mm plates or in 100 mm plates and were incubated at 37°C in 5% CO2 to

reach ~70% confluence before the experiments. The ARPE 19 cells were incubated with

either 0.2M or 2M of T4 for 1 hour before inducing ER stress by adding 500 M

palmitate (Sigma, Canada) in 0.5% BSA (Sigma, Canada). The cells were then incubated

at 37°C in 5% CO2 for 24 hours.

Cell viability

Cell viability was determined by annexin V and trypan blue staining. For Trypan Blue

exclusion, cells were seeded in 6 well plates (100,000 cells/well) and grown overnight.

The cells were treated with Tβ4 and palmitate as described before. After 24 hour

incubation, the cells were trypsinized and 100μl of the cell suspension was incubated with

an equal volume of 0.4% Trypan blue solution (Gibco, Canada) for 2 min. Cells were

counted using a hemocytometer on a light microscope, where live cells excluded Trypan

Blue staining. For annexin V staining, an annexin V staining kit (BD Biosciences) was

used. ARPE 19 cells were grown on coverslips in 35mm cell culture plates. The adherent

cells were incubated with annexin V and propidium iodide (PI) for 15 min. The cells were

fixed with 3% paraformaldehyde, washed with PBS and viewed under fluorescence

microscope with a dual filter set for FITC (Annexin V) and rhodamine (PI).

Intracellular ROS production

The dye H2DCFDA dye is used to detect intracellular ROS. ARPE 19 cells grown on

coverslips were incubated with H2DCFDA for 30 min at 37°C. The cells were washed

with PBS 3X and viewed under fluorescence microscope with excitation at 488nm and

emission at 530nm.

120

Cholesterol detection

Cholesterol estimation was done by using filipin (Sigma, Canada). For filipin staining, the

cells were fixed in 3% paraformaldehyde for 30 min and were then washed 3 times with

PBS. The fixed cells were incubated with 0.05mg/mL filipin (Sigma, Canada) for 1 hr in

dark. After incubation, the cells were washed 3X PBS and slides were mounted on slides

with fluoromount G (Southern Biotech). The slides were viewed under a fluorescent

microscope using a UV filter with excitation at 340-380 nm and emission at 385-470 nm.

To detect cholesterol, ARPE 19 cells were also stained with 750 nM PFO-D4-GFP for 30

min at 37 °C and 5% CO2. The cells were washed three times with HEPES buffer and

images were taken on an Axiovert fluorescence microscope with 535 nm/550 nm

excitation/emission.

Nitric oxide measurement

ARPE-19 cells were seeded at a density of 2X105 cells in a 10cm plate and were used

after they reached ~90% confluence. The cells were pretreated with Tβ4 for one hour and

then incubated in the presence of palmitate for 24 h. After the termination of the treatment

times, media was aspirated and fresh media was added. The cells were incubated again

and after 1 hr, 200 uL of media was withdrawn with a Hamilton gastight syringe and

directly injected into the purge vessel of a Sievers Nitric Oxide Analyzer (Model 280i)

which contained acetic acid and sodium iodide. A portion of NO produced by ARPE 19,

is oxidized by oxygen to form nitrite in the media. The acidified iodide in the purging

chamber of the NOA then reduces nitrite to NO. To further determine changes in NO

production, ARPE 19 cells were incubated with cell culture medium containing 5 M ,

4,5-diaminofluorescein diacetate (DAF-DA , Invitrogen) at 37°C in a 95% air/5% CO2

incubator for 30 min. Cells were then washed 3X with phosphate buffered saline (PBS)

and mounted on the stage of an Axiovert 200 inverted fluorescence microscope. DAF-DA

fluorescence was monitored using excitation and emission wavelengths of 485 and 538

nm, respectively.

Immunofluorescence

ARPE-19 cells were grown on coverslips in 35mm cell culture dishes. For the study of

nuclear translocation of NF-κB, cells were incubated with 500M palmitate for 24 hours,

121

with or without Tβ4 pretreatment. After incubation, the cells were washed with PBS and

fixed in ice cold ethanol for 10 min. Rabbit polyclonal antibody against the p65 subunit of

NFκB (1:200 dilution; Abcam) was used as the primary antibody. Alexa Fluor 488 -

labeled goat anti-rabbit IgG was used as a secondary antibody (1:500 dilution; Santa Cruz

Biotechnology). Preparations were mounted in Fluoromount G and examined by

fluorescence microscopy.

Nuclear and cytoplasmic fractions

After treatments, ARPE-19 cells were trypsinized, resuspended and homogenized in

buffer A (10 mM HEPES pH 7.8, 10 mM KCl, 1.5 mM MgCl2, 0.5% Nonidet P-40, 1

mM dithiothreitol (DTT) and protease inhibitor cocktail (Sigma)). Nuclei and cytosolic

fractions were separated by centrifugation at 1000g for 20 minutes. The cytosolic

fractions (supernatant) were stored at −80°C until further analysis. The nuclear fractions

(pellets) were resuspended in buffer B (5mM HEPES pH 7.8, 1.5mM MgCl2, 0.2mM

EDTA, 1mM DTT, 26% glycerol, 300mM NaCl pH 7.8 and protease inhibitor cocktail).

Nuclei were extracted for 30 min at 4°C. Soluble nuclear fractions were obtained by

centrifuging at 12,000g for 10 min. Supernatants (nuclear extracts) were stored at −80°C

until further NFB analysis. Protein concentration was determined by the BCA method.

Western blots

Proteins were extracted from ARPE 19 cells with lysis buffer (10g/L sodium

deoxycholate, 1% Triton X, 0.01% SDS, 150mM NaCl, 50mM Tris pH 7.5, 0.05 mM

EDTA, 50mM NaF, 10mM sodium pyrophosphate, 0.5mM sodium peroxyvanadate and

protease inhibitor cocktail). Protein concentration was determined by the BCA method.

10 μg of protein samples were resolved in 10% sodium dodecyl sulfate–polyacrylamide

gel and transferred onto polyvinylidene fluoride (PVDF) membranes. Membranes were

probed with primary antibodies against anti-rabbit eNOS (1:1000; Abcam), anti-rabbit

phospho-eNOS (1:1000; Cell Signalling), anti-rabbit p65 subunit of NFκB (1:1000;

Abcam), anti-rabbit Grp78 (1:1000; Abcam), anti-mouse PDI (1:1000, Abcam), anti-

rabbit Topo II (1:500; Santa Cruz) and anti-mouse actin (1:5000; Abcam). Membranes

were then incubated with HRP-conjugated anti-rabbit and anti-mouse secondary

122

antibodies (1:2000; Abcam) for 1 h and visualized using enhanced chemiluminescence

reagent (Pierce, USA).

Statistical analysis and images

Data is expressed as an average of all trials and statistical analysis was performed by

Student's t-test. The error bars represent standard deviation. The images were captured

with a Zeiss Axiovert fluorescence microscope and analyzed using Northern Eclipse.

123

RESULTS

Cell viability

The cells under ER stress for prolonged periods eventually undergo apoptosis. Apoptosis

is detected by the flipping of phosphatidylserine from the inner leaflet of the plasma

membrane to the outer leaflet. We determined the viability of the cells using FITC-

Annexin V, which binds to phosphatidylserine and detects apoptosis. PI was used to stain

the nucleus of viable cells. As shown in Fig. 1A, no apoptotic signal is observed in

controls and cells pretreated with 2 µM Tβ4. Very little annexin V binding is seen in cells

which were under ER stress (no Tβ4 pretreatment) and those pretreated with 0.2µM Tβ4.

The trypan blue exclusion assay showed that ~92% cells were viable in the controls;

however the cell viability decreased to ~78% in palmitate treated cells. The ARPE 19

cells which were pretreated with 0.2 µM and 2 µM Tβ4 had 80% and 82% viable cells

respectively (Fig. 1B). These results illustrated that after induction of ER stress for 24

hours, the cells are still viable and healthy and can be used for the subsequent studies.

124

Figure 1 – Detection of apoptosis and cell viability in ARPE 19 cells treated with

palmitate with or without Tβ4.

ARPE 19 cells were treated with 500 µM palmitate in the presence and absence of Tβ4

concentrations as indicated. (A) After 24 hours, cells were stained with PI (red) and

apoptotic cells were detected by Annexin V staining (green). Images were taken of a

minimum of five different fields in a slide using a 40X oil immersion objective. (B)

ARPE 19 cells were treated with palmitate and Tβ4 as described before. Live and dead

cells were determined using the trypan blue exclusion assay. The results are presented as

percentage of cell viability. Data is shown as mean ± S.D (n=4).

A

B

125

Induction of ER stress markers by palmitate

Grp78 and PDI are very important markers for ER stress induction. These molecular

chaperones are upregulated in response to ER stress. As shown in Figure 2, palmitate

treatment upregulated expression of Grp78 and PDI proteins which indicates induction of

ER stress. The Grp78 protein levels are brought down to normal (almost similar to

controls) when ARPE 19 cells were pretreated with 0.2 µM Tβ4 before inducing ER

stress.

Figure 2 – Expression of ER stress markers, Grp78 and PDI, in ARPE 19 cells

treated with palmitate in the presence or absence of T4.

ARPE 19 cells were treated with 500 µM palimate with or without Tβ4 for 24 hours.

Expression of Grp78 and PDI was determined from the cell lysates separated by SDS-

PAGE. The blots were probed with anti-rabbit Grp78 antibody and anti-mouse PDI

antibodies. Upper and middle panels show PDI and Grp78 expression in response to ER

stress and Tβ4 pretreatment and the lower panel shows actin, which was used as a loading

control. The graphs depict the blot densities determined by using Image J (n=3); * P <

0.05.

Control ERS ERS +

0.2µM TB4

PDI

Grp78

Actin

126

Tβ4 attenuates ER stress induced cholesterol production

ER stress is known to increase cholesterol levels in cells. Increased cholesterol

accumulation in RPE has also been associated with the pathogenesis of AMD (Curcio et

al 2005). The cholesterol determinations in ARPE-19 indicated that there was a ~4.5 fold

increase in cholesterol in cells under ER stress as compared to controls, however, treating

the cells with 0.2µM Tβ4 before ER stress induction significantly reduced cholesterol

levels by ~20%. Pretreatment with 2µM Tβ4 further decreases the cholesterol levels by

~27% (Figure 3).

127

A

B

128

Figure 3 – Effect of Tβ4 pretreatment on cholesterol in retinal pigment epithelial

cells under ER stress.

ARPE 19 cells were treated with palmitate for 24 hours, in the presence or absence of

Tβ4. (A) The cells fixed with 3% paraformaldehye were stained with filipin (blue) and PI

(red) and examined by fluorescence microscope. Filipin stains intracellular cholesterol

and PI stains the nucleus. The pictures were captured using 40X oil immersion objective

The graph depicts average fluorescence of filipin from four separate experiments (± SD);

*** P < 0.001. (B) Cholesterol was also estimated by staining with PFO-D4-GFP. After

treatment, the cells were incubated with PFO-D4-GFP at room temperature. After 30 min

incubation, the cells were fixed with 3% paraformaldehyde and visualized under

fluorescence microscope using 40 X oil immersion objective. The graph represents

average fluorescence of PFO-D4-GFP ± SD (n=4); *** P < 0.001.

129

Tβ4 pretreatment decreases ER stress induced ROS production

Oxidative stress is associated with the onset of ER stress and is one of the most important

factors associated with the progression of AMD. H2DCFA, a cell permeable dye which

incorporates into the hydrophobic regions of the cell, was used for this purpose. The

cellular esterases cleave the acetate moiety leaving impermeant, non-fluorescent, 2′,7′-

dichlorodihydrofluorescein (H2DCF), which is oxidized by reactive oxygen species to

fluorescent dichlorofluorescein (DCF). To determine whether Tβ4 has the ability to

reduce reactive oxygen species (ROS) formation after induction of ER stress, the levels of

ROS were determined. It can be clearly seen in Figure 4 that ER stress increased ROS

production by ~ 4 fold. Pretreatment with 0.2µM Tβ4 decreased ROS production by ~

42%. Higher concentration of Tβ4 (2µM) was able to reduce ROS formation by ~ 57%.

130

Figure 4 – Effect of Tβ4 pretreatment on intracellular ROS production in ER stress

induced retinal pigment epithelial cells.

ARPE 19 cells were incubated with 500 µM ER stressor palmitate, with or without Tβ4

(0.2μM or 2 µM) for 24 hours. The cells were analyzed for intracellular ROS using

H2DCFDA. The fluorescence (green) is generated by the oxidized product of H2DCFDA,

which indicates ROS formation. The graph shows an average of H2DCFDA fluorescence

and the results are an average of five different set of experiments (n=4); *** P < 0.001.

Pictures were taken using 63X objective.

0

10

20

30

40

50

60

70

80

90

Control ERS ERS+0.2uM TB4 ERS+2uM TB4

H2D

CF

DA

flu

ore

sc

en

ce ***

***

***

131

Tβ4 promotes nitric oxide production and eNOS phosphorylation

The palmitate treated cells were compared with the controls and Tβ4 pre treated cells with

respect to nitric oxide production. The nitric oxide was determined in the media, which is

an indirect method to analyze changes in nitric oxide production by cells. ER stressor

palmitate decreased nitric oxide production by ~ 55%, but NO production was

upregulated by ~ 88% in the cells which were treated with Tβ4 before inducing ER stress

as compared to cells under ER stress (Figure 5A). To further verify the role of Tβ4 in

nitric oxide production, eNOS expression and phosphorylation was determined. The

activity of eNOS is increased by its phosphorylation at Ser 1177. As illustrated in Figure

5C, there is no significant difference in the expression of eNOS in palmitate treated cells

as compared to the control and Tβ4 pretreated cells. eNOS phosphorylation decreased

after induction of ER stress by palmitate, whereas pretreatment with 0.2µM Tβ4

upregulated the phosphorylation of eNOS.

132

B

A

133

Figure 5 – Effect of Tβ4 on nitric oxide production, eNOS expression and

phosphorylation in retinal pigment epithelial cells under ER stress.

The ARPE 19 cells were treated with palmitate in the presence or absence of Tβ4 as

previously described. (A) After 24 hours, the cells were stained with 5µM DAF-DA for

30 min. The cells were washed with PBS and viewed under Zeiss Axiovert fluorescence

microscope using a 20X objective. The graph shows average intensity of DAF-DA

fluorescence from three independent experiments (± SD); * P < 0.05; ** P < 0.01. (B)

Total nitrite was measured for retinal pigment epithelial cells treated with palmitate for 24

hours with or without Tβ4. After treatment, the cell culture media was replaced with fresh

media and the cells were incubated again for 1 hour. The data shown is representative of

three different experiments (± SD); * P < 0.05; ** P < 0.01. (C) The expression of eNOS

and its phosphorylation at Ser 1177 was estimated from western blots using cell lysates of

control, cells under ER stress and cells treated with 0.2 µM Tβ4 before inducing ER

stress. The blots were probed with eNOS and Ser1177 phospho eNOS antibodies. Actin

was used as a loading control. The graph represents blot densities of eNOS and phospho

eNOS which were quantified using Image J software.

0

1000

2000

3000

4000

5000

6000

Control ERS ERS+0.2uM TB4

eN

OS

blo

t d

en

sit

y

C

134

Tβ4 reduces nuclear localization of NFκB

ER stress activates and induces the nuclear translocation of the transcription factor NFκB

(Pahl et al 1995; Kaneko et al 2003). As demostarted in Figure 6A, the

immunofluorescence staining of the p65 subunit of NFκB in control cells shows

cytoplasmic distribution. Incubation with ER stressor palmitate predominantly relocates

NFκB to the nucleus; however when the ER stressed cells were pretreated with 0.2 µM

and 2 µM Tβ4, this nuclear translocation was decreased, with large amounts of the p65

subunit being in the cytoplasm.

135

A

B

136

Figure 6 – NFκB localization in response to ER stress and T4 pretreatment.

(A) ARPE 19 cells were cultured on glass coverslips and treated with 500μM palmitate

with or without 0.2μM Tβ4 and 2μM Tβ4. The cells were fixed with ethanol and FITC

conjugated goat anti-rabbit secondary antibody was used to detect the p65 subunit of

NFκB. Nuclear localization of NFκB is shown by arrows. Images were taken at 40X oil

immersion objective. (B) Nuclear localization of NFκB was further detected by Western

blots of nuclear and cytoplasmic fractions. Relative levels of NFκB in nuclear fractions

were determined. There is a significant increase in nuclear NFκB protein in cells under

ER stress. Pretreatment with Tβ4 decreases NFκB localization to the nucleus in a

concentration dependent manner. Actin was used as a control for cytoplasmic fractions

and Topo II was used as a control for nuclear fractions.

137

Discussion

RPE dysfunction is one of the key factors involved in the progression of AMD. Retina

and RPE are under constant oxidative stress due to high metabolic activity, oxygen

consumption and light absorption. The fluctuations in the redox environment and ROS

production leads to disturbances in the ER hence causing ER stress (Lai E et al 2007). ER

stress has been proposed to be an important parameter involved in the pathogenesis of

AMD. A recent study has shown increased expression of tight junctions in retinal pigment

epithelial cells in vitro. This has been shown to alter the function of RPE cells which

might be involved in AMD (Yoshikawa et al 2011). However, not many studies have

foccussed on the effects of ER stress on RPE cells so far.

Tβ4 is a major actin sequestering protein which plays an important role in various

other physiological processes. It decreases cytochrome c release from the mitochondria

and also increases the expression of anti-apoptotic gene, bcl-2 (Sosne et al 2005).

Previous studies have illustrated the role of Tβ4 in decreasing inflammation (Sosne et al

2002) and preventing apoptosis (Ho et al 2010).

In the present study, we illustrated the effects of ER stress on retinal pigment

epithelial cells and the ability of Tβ4 to alleviate ER stress in RPE. ER stress disrupts

protein folding in the endoplasmic reticulum leading to accumulation of unfolded

proteins. An unfolded protein response (UPR) is initiated in order to maintain cell

homeostasis. UPR upregulates expression of chaperone proteins in order to augment the

protein folding capacity of the ER. When UPR is not able to alleviate ER stress, it

initiates an apoptotic signaling pathway, which involves JNK and caspases (Harding et al

2002; Kaufman et al 2002).

In this study, we evaluated the expression of molecular chaperones in retinal

pigment epithelial cells, in response to ER stress. Grp78 and PDI protein expression were

upregulated in the cells treated with palmitate, however, Tβ4 decreases the expression of

these chaperones (Fig. 2). The ability of Tβ4 to decrease Grp78 and PDI levels indicates

its potential role in protecting the cells against ER stress.

The role of oxidative stress in RPE dysfunction and AMD pathogenesis is very

well known. Previous reports have also linked oxidative stress with ER stress and RPE

dysfunction. The role of ER stress in RPE neovascularization in AMD has also been

138

implicated (He et al 2008; Libby et al 2010). It has been demonstrated in a previous study

that there is accumulation of free and esterified cholesterol in the RPE of AMD patients

(Lakkaraju et al 2007; Curcio et al 2005). The results of our study showed that Tβ4

pretreatment decreases cholesterol production, which accumulates as a result of ER stress

(Fig. 3). Oxidative stress, which occurs due to excessive ROS formation, plays a very

important role in RPE dysfunction. It is illustrated by our results that Tβ4 is able to

reduce intracellular ROS in RPE and hence protect the cells from oxidative damage.

We next determined the effect of Tβ4 on NO formation. Previous reports have

suggested that there is less production of nitric oxide in the eyes of AMD patients (Bhutto

et al 2010). In the present study, we demonstrate that Tβ4 is capable of upregulating NO

production in cells under ER stress, however, when levels of eNOS were determined,

there was no significant difference in its expression in treated versus untreated cells.

There was, however a significant difference in the phosphorylation of eNOS at Ser1177,

in the cells which were pretreated with Tβ4 before induction of ER stress (Fig 5). It has

been previously reported that Tβ4 can increase eNOS phosphorylation at Ser1177 in

endothelial progenitor cells (Qiu et al 2009). These results strongly suggest that Tβ4 is

capable of increasing eNOS phosphorylation and hence NO production in ARPE 19 cells.

The induction of ER stress is also associated with the activation of the

transcription factor NFκB. It is found in the cytoplasm but upon activation it translocates

to the nucleus, where it further promotes the transcription of various pro-inflammatory

genes and hence inflammatory signaling pathways. We further show that Tβ4 can also

decrease nuclear localization of NFκB (Fig. 6). Our observations agree with a previous

study done in corneal epithelial cells which showed that Tβ4 can minimize nuclear

localization of NFκB after TNF-α stimulation (Sosne et al 2007).

Conclusions

In summary, the present preliminary study demonstrates the protective effects of Tβ4 in

retinal pigment epithelial cells under ER stress. More work is required to be done in this

field to determine the protective effects of Tβ4 in AMD and other retinal diseases.

139

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Lai E, Teodoro T and Volchuk A. Endoplasmic reticulum stress: signalling the unfolded

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141

CHAPTER 6

General Discussion

142

Laminar shear stress is very critical for maintaining proper vascular hemostasis

functioning and hemostasis. From a clinical standpoint, shear stress is one of the most

potent endothelial stimulators and it plays an important role in numerous processes

including vasoregulation, chronic adaptive vessel remodeling

(Helmke et al 2002),

maintaining vascular homeostasis by inhibiting endothelial responses to cytokine

stimulation (Chiu et al 2002) and in the development of atherosclerosis. Previous studies

have shown that areas with low and oscillating shear stress are more prone to

atherosclerosis (Moore et al 1994; Pedersen et al 1999).

Platelet adhesion and thrombus development also depends on the local

hemodynamic environment to a large extent (Mustard et al 1966). Shear stress plays an

important role in transporting platelets and mediating their adhesion at the site of injury.

To understand the mechanisms responsible for platelet function under low and high shear

rates, many studies have focused on the platelet adhesion to various matrices (fibrinogen,

collagen) under flow conditions. In all of these studies, coating of glass slides with

fibrinogen, collagen or vWF has been reported and these flow devices were then used to

study platelet aggregation mechanisms (Neeves et al 2008; Fuchs et al 2010, Giesen et al

1999). This might result in heterogeneous coating and not all the surface might be coated.

To understand the role of physiological and pathological shear stress in various

vascular diseases, many flow devices have been engineered (Andrews et al 2010; Man et

al 2009; Shepherd et al 2009). These flow devices require a large population of

endothelial cells which needs more chemicals and reagents. Recently, many studies have

developed microfluidic flow chambers which are very costly. Out of the various flow

devices available to study effects of shear stress, parallel plate flow chambers are the

most common. Parallel plate flow chambers are used to mimic flow conditions occurring

in vivo and study the dynamic response of vascular endothelial cells and platelets to

controlled levels of fluid shear stress.

This thesis describes the development and validation of flow devices to study

effects of shear stress on endothelial cells and platelet aggregation. The parallel plate flow

chamber described in here can be used to study shear mediated effects on endothelial cells

and platelets. These flow devices are very versatile and economical to make and allow

real time study of the platelets and endothelial cells exposed to shear stress. In the present

143

study, I report the construction of another flow device for studying platelet function, in

whole blood, in real time. In these flow chambers, the inert polydimethylsiloxane

(PDMS) surface was plasma-activated and a homobifunctional cross-linker was used to

immobilize platelet-binding proteins onto its surface. The current study introduces a new

facile and robust method for the construction flow chambers which enable the kinetic

monitoring of platelet adhesion in whole blood.

Type 2 diabetic patients are at a greater risk of developing vascular diseases like

atherosclerosis. Increased levels of fibrinogen have been reported in diabetes, which

might contribute to abnormal clot formation in diabetic patients (Banga et al, 1986).

Plasmin activator inhibitor 1 (PAI 1) inhibits the formation of plasmin from plasminogen.

Increased levels of this inhibitor have also been found in the diabetic blood (Juhan-Vague

et al, 1989). Previous studies have also shown increased levels of vWF activity in

diabetes (Van Zile J et al 1981). All of these factors contribute to the formation of a

prothrombotic state in the diabetic patients.

The flow chamber developed in this thesis was used to study platelet aggregation

in diabetic subjects in vitro. The flow chamber was able to discern differences between

platelets from normal and diabetic subjects. These flow devices provide real time data on

the kinetics of platelet binding and can detect subtle pathological changes in platelet

function introduced as a result of pathologies such as type 2 diabetes.

Tβ4 is a 43 amino acid peptide found in various tissues and cells including

platelets. It has been implicated in the sequestering of G actin in mammalian cells. Tβ4

plays an important role in preventing apoptosis, corneal wound healing, cell survival and

angiogenesis (Sosne et al 2004; Hinkel et al 2008; Smart et al 2007).

The aim of this work was to evaluate the role of Tβ4 in various physiological

processes. It has been shown previously that Tβ4 is secreted by platelets and is

crosslinked by factor XIIIa (a transglutaminase) to fibrin in a time- and Ca2+-

dependent

manner (Huff et al 2002; Makogoneko et al 2004). This increases the local concentration

of Tβ4 near sites of clots and tissue damage, where it is postulated to contribute to wound

healing, angiogenesis and inflammatory responses. Yet the role of Tβ4 on platelet

thrombus formation has yet to be fully investigated.

The current study represents a preliminary effort towards this goal. Here, we have

144

tested the effects of Tβ4 extraneously added to whole blood, on deposition of ADP

activated platelets onto fibrinogen under conditions of continuous flow and shear. There

was an increase in platelet deposition when Tβ4 was added to the blood, as compared to

the controls. However, at high concentrations of Tβ4, a decrease in the platelet deposition

was observed. The results of the present study suggest that there could be a high affinity

Tβ4-binding domain on fibrinogen. The fibrinogen binding ability of Tβ4 could explain

the biphasic response of Tβ4 on platelet deposition under conditions of flow as observed

here. Furthermore, the results obtained in this study suggest a key modulatory role for

Tβ4 in thrombus formation. At low doses Tβ4 promotes platelet deposition and

aggregation by as yet unknown mechanism(s). But as platelets are activated and more

Tβ4 is released from platelets, its serum levels rises and prevent excessive thrombus

formation by binding to fibrinogen and preventing its interaction with platelets.

Tβ4 has also been shown to decrease inflammation and promote corneal wound

healing, and various studies have given evidence supporting the role of Tβ4 in cornea

protection (Ho et al 2008; Sosne et al 2007; Dunn et al 2010). This thesis also

investigates the effects of Tβ4 on endoplasmic reticulum stress in retinal pigment

epithelial cells. In this study, we illustrated that Tβ4 is able to reduce ROS production,

which is an important factor associated with the progression of various ophthalmologic

diseases including AMD and DR (He et al 2008; Libby et al 2010). Tβ4 also promotes

NO production and decreases NF-κB nuclear localization in RPE. Our observations agree

with a previous study done in corneal epithelial cells which showed that Tβ4 can

minimize nuclear localization of NFκB after TNF-α stimulation (Sosne et al 2007). The

results reported in this thesis demonstrated that Tβ4 can mitigate the effects of ER stress

in RPE cells.

Overall, the work done in this thesis has done significant contributions in

understanding the role of Tβ4 in thrombosis. However, more work is needed in this field

to fully understand the role of Tβ4 in thrombosis and evaluate the mechanisms and

signalling pathways involved in this process. Very few studies have reported any role of

ER stress in retinal diseases and this is one of those few studies. This thesis also presents

preliminary data which demonstrates the protective effects of Tβ4 in retinal pigment

epithelial cells under ER stress. More extensive study needs to be done in future in this

145

field to get better understanding of the mechanisms involved. In conclusion, the work

presented in this thesis has made major contributions in development of flow devices to

study thrombosis and endothelial cell function under flow. This work is also a very

important step in studying the role of Tβ4 in various physiological processes.

146

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Oxide 2010; 23(4):335-42.

Banga JD and Sixma JJ. Diabetes mellitus, vascular disease and thrombosis.Clin

Haematol 1986; 15(2): 465-492.

Chiu JJ, Lee PL, Lee CI, Chen LJ, Chen CN, Ko YC, Lien SC. Shear stress attenuates

tumor necrosis factor-alpha-induced monocyte chemotactic protein-1 expressions

in endothelial cells. Clin J Physiol 2002; 45(4):169-76.

Dunn SP, Heidemann DG, Chow CY, Crockford D, Turjman N, Angel J, Allan CB,

Sosne G. Treatment of chronic nonhealing neurotrophic corneal epithelial defects

with thymosin beta 4. Arch Ophthalmol 2010; 128(5):636-8.

Fuchs B, Budde U, Schulz A, Kessler CM, Fisseau C and Kannicht C. Flow-based

measurements of von Willebrand factor (VWF) function: Binding to collagen and

platelet adhesion under physiological shear rate. Thrombosis Research 2010; 125

(3): 239-245.

Giesen PLA , Ursula Rauch U,

Bohrmann B,

Kling D, Roqué M, Fallon JT,

Badimon JJ,

Himber J, Riederer MA and Nemerson Y.

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He S, Yaung J, Kim YH, Barron E, Ryan SJ and Hinton DR. Endoplasmic reticulum

stress induced by oxidative stress in retinal pigment epithelial cells. Graefes Arch

Clin Exp Ophthalmol 2008: 246(5); 677-683.

Helmke BP, Davies PF. The cytoskeleton under external fluid mechanical forces:

hemodynamic forces acting on the endothelium. Ann Biomed Eng. 2002;

30(3):284-96.

Hinkel R, El-Aouni C, Olson T, Horstkotte J, Mayer S, Müller S, Willhauck M, Spitzweg

C, Gildehaus FJ, Münzing W, Hannappel E, Bock-Marquette I, DiMaio JM,

Hatzopoulos AK, Boekstegers P and Kupatt C. Thymosin beta 4 is an essential

paracrine factor of embryonic endothelial progenitor cell-mediated

cardioprotection. Circulation 2008; 117: 2232–2240.

Ho JH, Tseng KC, Ma WH, Chen KH, Lee OK, Su Y. Thymosin beta-4 upregulates anti

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fibrin and collagen. FASEB J 2002; 16: 691–696.

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plasminogen activator inhibitor activity in non-insulin-dependent diabetic

patients: relationship with plasma insulin. Thromb Haemost 1989; 61:370–373.

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Libby RT and Gould DB. Endoplasmic reticulum stress as a primary pathogenic

mechanism leading to age-related macular degeneration. Adv Exp Med Biol 2010;

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Makogonenko E, Goldstein AL, Bishop PD and Medved L. Factor XIIIa incorporates

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148

APPENDIX

The results presented in this section of the thesis relate to the studies in Chapter 2 of this

thesis.

Background

Earlier it was assumed that in parallel plate flow chambers, all cells are subjected to the

same average shear stress. However recently it has been hypothesized that cells are

subjected to variable shear stress depending on the area in the flow chamber (McCann et

al 2005). The objective of this study is to develop a flow device to study effects of shear

stress on endothelial cells and also to develop computational fluid dynamic (CFD)

template with different shear rates. The templates are designed to match the geometry of a

custom built in vitro flow cell device and define several regions of shear stress to which

the cells are exposed during in vitro experiment. Since it has previously been reported

that shear stress is correlated to NO production in endothelial cells, NO was measured in

order to validate the proposed flow device and the shear stress template.

Experimental Methods

Cell Culture

Human dermal microvascular endothelial cells (HDMECs) were purchased from

ScienCell. The cells were cultured in endothelial cell media (ECM, ScienCell) containing

10% fetal bovine serum (FBS, ScienCell), pencillin-streptomycin (ScienCell) and

endothelial cell growth supplement (ECGS, ScienCell) at 37ºC in a 5% CO2 incubator.

The flow chambers were coated 0.1% fibronectin overnight and endothelial cells were

grown until 95% confluent.

In vitro set-up

The flow cell circuit used for this investigation consists of a custom designed flow cell,

sterile tubing, a medium reservoir, and a peristaltic pump. The entire set-up was kept at

37ºC in a 5% CO2 incubator. The cells were exposed to shear stress for 12 hours.

149

Fluorescence Indicator (DAF-2)

To determine changes in NO production, endothelial cell monolayers were incubated with

cell culture medium containing 5 M, 4,5-diaminofluorescein diacetate (DAF-DA ,

Invitrogen) at 37°C in a 95% air/5% CO2 incubator for 30 min. In the presence of oxygen,

DAF-2 reacts with NO to yield the highly fluorescent triazolofluorescein (DAF-2T). The

fluorescence was monitored using excitation and emission wavelengths of 485 and 538

nm, respectively.

Results

Geometry of the flow cell

The flow cell has the dimensions shown in Figure 2. The total area on the flow cell

surface available for cell growth and attachment was 6.85 x 10-5

m2. The walls of the flow

cell are made of PDMS (Dow Corning) and the cells are grown on a glass coverslip.

Figure 1 – Geometry of the custom flow cell.

150

Velocity gradient template

To simulate the flow in the chamber and estimate the shear stress for the testing program,

a computational model of the flow channel was developed.

1mL/min

Figure 2 – Velocity gradient template.

151

Endothelial morphology in response to shear stress

The human dermal microvascular endothelial cells were exposed to laminar shear stress

for 12 hours hours and morphological changes were observed using light microscope. The

cells become elongated and aligned in the direction of flow; however no such change was

observed in endothelial cells maintained under static conditions.

Figure 3 – Morphology of endothelial cells grown under static and laminar shear

stress conditions.

Effect of shear stress on NOx production

In order to validate the flow chamber can be employed for studies in the areas of

biomedical research; the effect of fluid shear stress on nitric oxide production in

endothelial cells was examined. NOx formation was upregulated when cells were grown

under shear as compared to static conditions. The increase in NOx production was more

in endothelial cells which were exposed to high shear stress for 12 hours, compared with

those under low shear (Fig. 3).

152

0.344-0.451 Pa 0.236-0.344 Pa 0.129-0.236 Pa 0.021-0.129 Pa

Figure 4 – NOx upregulation in response to shear stress.

HDMECs were exposed to shear stress for 12 hours and then stained with 5 μM DAF-

DA. The regions exposed to high shear stress had increased NO production. The graph

depicts the average fluorescence intensity of DAF-DA (± SD).

0

10

20

30

40

50

60

0.021 -0.129 Pa

0.129 -0.236 Pa

0.236 -0.344 Pa

0.344 -0.451 Pa

DA

F-D

A flu

ore

sc

en

ce

Static

Shear stress

Shear

stress

Static

153

Discussion

Shear stress plays a critical role in maintaining vascular hemostasis and also in the

pathogenesis of various diseases, particularly atherosclerosis. Numerous studies have

focussed on investigating the effects of shear stress on endothelial cells. Parallel plate

flow chambers are commonly used for this purpose.

In this study, we describe the development and validation of a flow chamber for

shear stress studies. It has previously been shown that the endothelial cells align in the

direction of the flow and that the alignment would be more pronounced at areas of higher

shear. Our results also agree with these studies and show similar results.

There was an increase in NO production in regions of high shear. These findings

are in agreement with the previous studies which reported NO upregulation in response to

shear stress. These results suggest that this chamber can be used to study shear stress

effects on endothelial cells. This chamber is very simple to make, convenient to use and

inexpensive. It can be re used and requires very less number of cells. The shear stress

template described in here, can be used to define different regions of known shear stress.

This allows simultaneous exposure to different shear stress levels within one flow cell

under the exact same experimental conditions.

References

McCann JA, Peterson SD, Plesniak MW, Webstre TJ and Haberstroh KM. Non-uniform

flow behavior in a parallel plate flow chamber alters endothelial cell responses. Annals of

Biomedical Engineering 2005; 33 (3): 2005328–2005336.

154

VITA AUCTORIS

Name: Harmanpreet Kaur

Place of Birth: Ludhiana, Punjab, India

Year of Birth: 1981

Education: Panjab University

Chandigarh, India

2000 – 2003, B.Sc

Panjab University

Chandigarh, India

2003 – 2005, M.Sc Honors

University of Windsor

Windsor, Ontario, Canada

2006 – 2011, PhD Biochemistry